PYRROLO [3, 4-h] ISOQUINOLINE COMPOUNDS AND METHODS FOR MODULATING GATED ION CHANNELS

ABSTRACT

The present invention relates to compositions and methods to modulate the activity of gated ion channels.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/603,946, Attorney Docket No. PCI-034RCE, filed Nov. 22, 2006,entitled PYRROLO [3,4-h]ISOQUINOLINE COMPOUNDS AND METHODS FORMODULATING GATED ION CHANNELS (as amended), which claims priority toU.S. Provisional Application No. 60/739,600, Attorney Docket No.PCI-034-1, filed Nov. 23, 2005, entitled “COMPOSITIONS AND METHODS FORMODULATING GATED ION CHANNELS.” The contents of any patents, patentapplications, and references cited throughout this specification arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to compositions which modulate theactivity of gated ion channels and methods and uses thereof.

BACKGROUND

Mammalian cell membranes are important to the structural integrity andactivity of many cells and tissues. Of particular interest is the studyof trans-membrane gated ion channels which act to directly andindirectly control a variety of pharmacological, physiological, andcellular processes. Numerous gated ion channels have been identified andinvestigated to determine their roles in cell function.

Gated ion channels are involved in receiving, integrating, transducing,conducting, and transmitting signals in a cell, e.g., a neuronal ormuscle cell. Gated ion channels can determine membrane excitability.Gated ion channels can also influence the resting potential ofmembranes, wave forms, and frequencies of action potentials, andthresholds of excitation. Gated ion channels are typically expressed inelectrically excitable cells, e.g., neuronal cells, and are multimeric.Gated ion channels can also be found in nonexcitable cells (e.g.,adipose cells or liver cells), where they can play a role in, forexample, signal transduction.

Among the numerous gated ion channels identified to date are channelsthat are responsive to, for example, modulation of voltage, temperature,chemical environment, pH, ligand concentration and/or mechanicalstimulation. Examples of specific modulators include: ATP, capsaicin,neurotransmitters (e.g., acetylcholine), ions, e.g., Na⁺, Ca⁺, K⁺, H⁺,Zn⁺, Cd⁺, and/or peptides, e.g., FMRF. Examples of gated ion channelsresponsive to these stimuli are members of the DEG/ENaC, TRPV and P2Xgene superfamilies.

Members of the DEG/ENaC gene superfamily show a high degree offunctional heterogeneity with a wide tissue distribution that includestransporting epithelia as well as neuronal excitable tissues. DEG/ENaCproteins are membrane proteins which are characterized by twotransmembrane spanning domains, intracellular N- and C-termini and acysteine-rich extracellular loop. Depending on their function in thecell, DEG/ENaC channels are either constitutively active like epithelialsodium channels to (ENaC) which are involved in sodium homeostasis, oractivated by mechanical stimuli as postulated for C. elegans degnerins,or by ligands such as peptides as is the case for FaNaC from Helixaspersa which is a FMRF amide peptide-activated channel and is involvedin neurotransmission, or by protons as in the case for the acid sensingion channels (ASICs). The mammalian members of this gene family known todate are αENaC (also known as SCNN1A or scnn1A), βENaC (also known asSCNN1B or scnn1B), γENaC (also known as SCNN1G or scnn1G), δENaC (alsoknown as ENaCd, SCNN1D, scnn1D and dNaCh), ASIC1a (also known as ASIC,ASIC1, BNaC2, hBNaC2, ASICalpha, ACCN2 and Accn2), ASIC1b (also known asASICbeta), ASIC2a (also known as BNC1, MDEG1, BNaC1 and ACCN1), ASIC2b(also known as MDEG2, ASIC2b), ASIC3 (also known as hASIC3, DRASIC,TNaC1, SLNAC1, ACCN3 and Accn3), ASIC4 (also known as BNaC4, SPASIC,ACCN4 and Accn4), BLINaC (also known as hINaC, ACCN5 and Accn5), andhINaC. For a recent review on this gene superfamily see Kellenberger, S,and Schild, L. (2002) Physiol. Rev. 82:735, incorporated herein byreference.

There are seven presently known members of the P2X gene superfamily; P2X(also known as P2RX1), P2X₂ (also known as P2RX2), P2X₃ (also known asP2RX3), P2X₄ (also known as P2RX4), P2X₅ (also known as P2RX5), P2X₆(also known as P2RX6), and P2X₇ (also known as P2RX7). P2X proteinstructure is similar to ASIC protein structure in that they contain twotransmembrane spanning domains, intracellular N- and C-termini and acysteine-rich extracellular loop. All P2X receptors open in response tothe release of extracellular ATP and are permeable to small ions andsome have significant calcium permeability. P2X receptors are abundantlydistributed on neurons, glia, epithelial, endothelia, bone, muscle andhematopoietic tissues. For a recent review on this gene superfamily, seeNorth, R. A. (2002) Physiol. Rev. 82:1013, incorporated herein byreference.

The receptor expressed in sensory neurons that reacts to the pungentingredient in chili peppers to produce a burning pain is the capsaicin(TRPV or vanilloid) receptor, denoted TRPV1 (also known as VR1,TRPV1alpha, TRPV1beta). The TRPV 1 receptor forms a nonselective cationchannel that is activated by capsaicin and resiniferatoxin (RTX) as wellas noxious heat (>43° C.), with the evoked responses potentiated byprotons, e.g. H⁺ ions. Acid pH is also capable of inducing a slowlyinactivating current that resembles the native proton-sensitive currentin dorsal root to ganglia. Expression of TRPV1, although predominantlyin primary sensory neurons, is also found in various brain nuclei andthe spinal cord (Physiol. Genomics 4:165-174, 2001).

Two structurally related receptors, TRPV2 (also known as VRL1 and VRL)and TRPV4 (also known as VRL-2, Trp12, VROAC, OTRPC4), do not respond tocapsaicin, acid or moderate heat but rather are activated by hightemperatures (Caterina, M. J., et al. (1999) Nature. 398(6726):436-41).In addition, this family of receptors, e.g., the TRPV or vanilloidfamily, contains the ECAC-1 (also known as TRPV5 and CAT2, CaT2) andECAC-2 (also known as TRPV6, CaT, ECaC, CAT1, CATL, and OTRPC3)receptors which are calcium selective channels (Peng, J. B., et al.(2001) Genomics 76(1-3):99-109). For a recent review of TRPV (vanilloid)receptors, see Szallasi, A. and Blumberg, P. M. (1999) Pharmacol. Rev.51:159, incorporated herein by reference.

The ability of the members of the gated ion channels to respond tovarious stimuli, for example, chemical (e.g., ions), thermal andmechanical stimuli, and their location throughout the body, e.g., smalldiameter primary sensory neurons in the dorsal root ganglia andtrigeminal ganglia, as well data derived from in vitro and in vivomodels has implicated these channels in numerous neurological diseases,disorders and conditions. For example, it has been shown that the ratASIC2a channel is activated by the same mutations as those causingneuronal degeneration in C. elegans. In addition, these receptors areactivated by increases in extracellular proton, e.g., H⁺, concentration.By infusing low pH solutions into skin or muscle as well as prolongedintradermal infusion of low pH solutions creates a change inextracellular pH that mimics the hyperalgesia of chronic pain.Furthermore, transgenic mice, e.g., ASIC2a, ASIC3, P2X₃ transgenic mice,all have modified responses to noxious and non-noxious stimuli. Thus,the biophysical, anatomical and pharmacological properties of the gatedion channels are consistent with their involvement in nociception.

Research has shown that ASICs play a role in pain, neurological diseasesand disorders, gastrointestinal diseases and disorders, genitourinarydiseases and disorders, and inflammation. For example, it has been shownthat ASICs play a role in pain sensation (Price, M. P. et al., Neuron.2001; 32(6): 1071-83; Chen, C. C. et al., Neurobiology 2002; 99(13)8992-8997), including visceral and somatic pain (Aziz, Q. Eur. J.Gastroenterol. Hepatol. 2001; 13(8):891-6); chest pain that accompaniescardiac ischemia (Sutherland. S. P. et al. (2001) Proc Natl Acad Sci USA98:711-716), and chronic hyperalgesia (Sluka, K. A. et al., Pain. 2003;106(3):229-39). ASICs in central neurons have been shown to possiblycontribute to the neuronal cell death associated with brain ischemia andepilepsy (Chester, M., Physiol. Rev. 2003; 83: 1183-1221; Lipton, P.,Physiol. Rev. 1999; 79:1431-1568). ASICs have also been shown tocontribute to the neural mechanisms of fear conditioning, synapticplasticity, learning, and memory (Wemmie, J. et al., J. Neurosci. 2003;23(13):5496-5502; Wemmie, J. et al., Neuron. 2002; 34(3):463-77). ASICshave been shown to be involved in inflammation-related persistent painand inflamed intestine (Wu, L. J. et al., J. Biol. Chem. 2004;279(42):43716-24; Yiangou, Y., et al., Eur. J. Gastroenterol. Hepatol.2001; 13(8): 891-6), and gastrointestinal stasis (Holzer, Curr. Opin.Pharm. 2003; 3: 618-325). Recent studies done in humans indicate thatASICs are the primary sensors of acid-induced pain (Ugawa et al., J.Clin. Invest. 2002; 110: 1185-90; Jones et al., J. Neurosci. 2004; 24:10974-9). Furthermore, ASICs are also thought to play a role ingametogenesis and early embryonic development in Drosophila (Darboux, I.et al., J. Biol. Chem. 1998; 273(16):9424-9), underlie mechanosensoryfunction in the gut (Page, A. J. et al. Gastroenterology. 2004;127(6):1739-47), and have been shown to be involved in endocrine glands(Grunder, S. et al., Neuroreport. 2000; 11(8): 1607-11). Therefore,compounds that modulate these gated ion channels would be useful in thetreatment of such diseases and disorders.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound of the Formula 1,Formula 2, Formula 3, Formula 4 or Formula 5, as well as a compoundselected from the group consisting of5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(3,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(3,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(2,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-phenyl-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(2,3-dimethyl-phenyl)-8-ethyl-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime;8-ethyl-5-(2-methoxy-phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime; and5-(2-ethoxy-phenyl)-8-ethyl-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime.

In one aspect, the invention provides a method of modulating theactivity of a gated ion channel, comprising contacting a cell expressinga gated ion channel with an effective amount of a compound of theinvention

In another embodiment of the invention, contacting the cells with aneffective amount of a compound of the invention inhibits the activity ofthe gated ion channel. In yet another embodiment, the gated ion channelis comprised of at least one subunit selected from the group consistingof a member of the DEG/ENaC, P2X, and TRPV gene superfamilies. In stillanother embodiment, the gated ion channel is comprised of at least onesubunit selected from the group consisting of αENaC, βENaC, γENaC,δENaC, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, BLINaC, hINaC,P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, P2X₇, TRPV1, TRPV2, TRPV3, TRPV4,TRPV5, and TRPV6. In another embodiment, the gated ion channel ishomomultimeric. In still another embodiment, the gated ion channel isheteromultimeric. In yet another embodiment, the DEG/ENaC gated ionchannel is comprised of at least one subunit selected from the groupconsisting of αENaC, βENaC, γENaC, δENaC, BLINaC, hINaC, ASIC1a, ASIC1b,ASIC2a, ASIC2b, ASIC3, and ASIC4. In another embodiment, the DEG/ENaCgated ion channel is comprised of at least one subunit selected from thegroup consisting of ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. Instill another embodiment, the gated ion channel comprises ASIC1a and/orASIC3. In yet another embodiment, the P2X gated ion channel comprises atleast one subunit selected from the group consisting of P2X₁, P2X₃,P2X₄, P2X₅, P2X₆, and P2X₇. In another embodiment, the TRPV gated ionchannel comprises at least one subunit selected from the group TRPV1,TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6. In still another embodiment, theheteromultimeric gated ion channels include the following combinationsof gated ion channels: αENaC, βENaC and γENaC; αENaC, βENaC and δENaC;ASIC1a and ASIC3; ASIC1b and ASIC3; ASIC2a and ASIC3; ASIC2b and ASIC3;ASIC1a, ASIC2a and ASIC3; P2X_(i) and P2X₂; P2X₁ and P2X₅; P2X₂ andP2X₃; P2X₂ and P2X₆; P2X₄ and P2X₆; TRPV1 and TRPV2; TRPV5 and TRPV6;and TRPV1 and TRPV4. In yet another embodiment, the heteromultimericgated ion channels include the following combinations of gated ionchannels: ASIC1a and ASIC2a; ASIC2a and ASIC2b; ASIC1b and ASIC3; andASIC3 and ASIC2b.

In another embodiment of the invention, the activity of the gated ionchannel is associated with pain. In yet another embodiment, the activityof the gated ion channel is associated with an inflammatory disorder. Instill another embodiment, the activity of the gated ion channel isassociated with a neurological disorder.

In another embodiment, the pain is selected from the group consisting ofcutaneous pain, somatic pain, visceral pain and neuropathic pain. Instill another embodiment, the pain is acute pain or chronic pain. In yetanother embodiment, the cutaneous pain is associated with injury,trauma, a cut, a laceration, a puncture, a burn, a surgical incision, aninfection or acute inflammation. In another embodiment, the somatic painis associated with an injury, disease or disorder of the musculoskeletaland connective system. In still another embodiment, the injury, diseaseor disorder is selected from the group consisting of sprains, brokenbones, arthritis, psoriasis, eczema, and ischemic heart disease. In yetanother embodiment, the visceral pain is associated with an injury,disease or disorder of the circulatory system, the respiratory system,the gastrointestinal system, or the genitourinary system. In anotherembodiment, the disease or disorder of the circulatory system isselected from the group consisting of ischaemic heart disease, angina,acute myocardial infarction, cardiac arrhythmia, phlebitis, intermittentclaudication, varicose veins and hemorrhoids. In still anotherembodiment, the disease or disorder of the respiratory system isselected from the group consisting of asthma, respiratory infection,chronic bronchitis and emphysema. In yet another embodiment, the diseaseor disorder of the gastrointestinal system is selected from the groupconsisting of gastritis, duodenitis, irritable bowel syndrome, colitis,Crohn's disease, gastrointestinal reflux disease, ulcers anddiverticulitis.

In another embodiment, the disease or disorder of the genitourinarysystem is selected from the group consisting of cystitis, urinary tractinfections, glomuerulonephritis, polycystic kidney disease, kidneystones and cancers of the genitourinary system. In still anotherembodiment, the somatic pain is selected from the group consisting ofarthralgia, myalgia, chronic lower back pain, phantom limb pain,cancer-associated pain, dental pain, fibromyalgia, idiopathic paindisorder, chronic non-specific pain, chronic pelvic pain, post-operativepain, and referred pain. In yet another embodiment, the neuropathic painis associated with an injury, disease or disorder of the nervous system.In another embodiment, the injury, disease or disorder of the nervoussystem is selected from the group consisting of neuralgia, neuropathy,headache, migraine, psychogenic pain, chronic cephalic pain and spinalcord injury.

In another embodiment of the invention, the activity of the gated ionchannel is selected from an inflammatory disorder of the musculoskeletaland connective tissue system, the respiratory system, the circulatorysystem, the genitourinary system, the gastrointestinal system or thenervous system. In another embodiment, the inflammatory disorder of themusculoskeletal and connective tissue system is selected from the groupconsisting of arthritis, psoriasis, myocitis, dermatitis and eczema. Instill another embodiment, the inflammatory disorder of the respiratorysystem is selected from the group consisting of asthma, bronchitis,sinusitis, pharyngitis, laryngitis, tracheitis, rhinitis, cysticfibrosis, respiratory infection and acute respiratory distress syndrome.In yet another embodiment, the inflammatory disorder of the circulatorysystem is selected from the group consisting of vasculitis, haematuriasyndrome, artherosclerosis, arteritis, phlebitis, carditis and coronaryheart disease. In another embodiment, the inflammatory disorder of thegastrointestinal system is selected from the group consisting ofinflammatory bowel disorder, ulcerative colitis, Crohn's disease,diverticulitis, viral infection, bacterial infection, peptic ulcer,chronic hepatitis, gingivitis, periodentitis, stomatitis, gastritis andgastrointestinal reflux disease. In still another embodiment, theinflammatory disorder of the genitourinary system is selected from thegroup consisting of cystitis, polycystic kidney disease, nephriticsyndrome, urinary tract infection, cystinosis, prostatitis, salpingitis,endometriosis and genitourinary cancer.

In another embodiment, the neurological disorder is selected from thegroup consisting of schizophrenia, bipolar disorder, depression,Alzheimer's disease, epilepsy, multiple sclerosis, amyotrophic lateralsclerosis, stroke, addiction, cerebral ischemia, neuropathy, retinalpigment degeneration, glaucoma, cardiac arrhythmia, shingles,Huntington's chorea, Parkinson disease, anxiety disorders, panicdisorders, phobias, anxiety hysteria, generalized anxiety disorder, andneurosis.

In another aspect, the invention provides a method of treating pain in asubject in need thereof, comprising administering to the subject aneffective amount of a compound of the invention. In one embodiment, thesubject is a mammal. In still another embodiment, the mammal is a human.

In yet another embodiment, the pain is selected from the groupconsisting of cutaneous pain, somatic pain, visceral pain andneuropathic pain. In another embodiment, the pain is acute pain orchronic pain.

In another aspect, the invention provides a method of treating aninflammatory disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of a compound of theinvention. In one embodiment, the subject is a mammal. In still anotherembodiment, the mammal is a human.

In yet another embodiment, the inflammatory disorder is an inflammatorydisorder of the musculoskeletal and connective tissue system, therespiratory system, the circulatory system, the genitourinary system,the gastrointestinal system or the nervous system.

In another aspect, the invention provides a method of treating aneurological disorder in a subject in need thereof, comprisingadministering an effective amount of a compound of the invention. In oneembodiment, the subject is a mammal. In still another embodiment, themammal is a human.

In yet another embodiment, the neurological disorder is selected fromthe group consisting of schizophrenia, bipolar disorder, depression,Alzheimer's disease, epilepsy, multiple sclerosis, amyotrophic lateralsclerosis, stroke, addiction, cerebral ischemia, neuropathy, retinalpigment degeneration, glaucoma, cardiac arrhythmia, shingles,Huntington's chorea, Parkinson disease, anxiety disorders, panicdisorders, phobias, anxiety hysteria, generalized anxiety disorder, andneurosis.

In another aspect, the invention provides a method of treating a diseaseor disorder associated with the genitourinary and/or gastrointestinalsystems of a subject in need thereof, comprising administering to thesubject an effective amount of a compound of the invention. In anotherembodiment, the subject is a mammal. In still another embodiment, themammal is a human.

In yet another embodiment the disease or disorder of thegastrointestinal system is selected from the group consisting ofgastritis, duodenitis, irritable bowel syndrome, colitis, Crohn'sdisease, ulcers and diverticulitis. In another embodiment, the diseaseor disorder of the genitourinary system is selected from the groupconsisting of cystitis, urinary tract infections, glomuerulonephritis,polycystic kidney disease, kidney stones and cancers of thegenitourinary system.

In another embodiment of the invention, the methods further compriseadministering an adjuvant composition. In yet another embodiment, theadjuvant composition is selected from the group consisting of opioidanalgesics, non-opioid analgesics, local anesthetics, corticosteroids,non-steroidal anti-inflammatory drugs, non-selective COX inhibitors,non-selective COX2 inhibitors, selective COX2 inhibitors,antiepileptics, barbiturates, antidepressants, marijuana, and topicalanalgesics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C illustrate dose-response curves of the inhibitoryeffect of compounds E, F and G on hASIC1a activity as described inExample 1. HEK293 cells were transfected with hASIC1a and cells wereexposed to acidic buffer in the absence and presence of increasingconcentrations of Compounds E, F or G. Gated-channel activity wasdetermined by measuring intracellular calcium variation using acalcium-selective fluorescent dye. Compounds E, F and G dose-dependentlyinhibit acid-induced hASIC1a activity.

FIGS. 2A and 2B illustrate dose-response curves of the inhibitory effectof Compounds A and H on acid-induced peak inward currents elicited inHEK293 transfected with rat ASIC1a or rat ASIC3, as described in Example2. Acid-induced inward currents were recorded in the presence andabsence of increasing concentrations of Compound A or Compound H usingthe whole-cell configuration of the patch-clamp method (voltage clampmode). Compounds A and H dose-dependently inhibit acid-induced rASIC1aand rASIC3 activity.

FIG. 3 illustrates the inhibitory effect of Compound A on acid-evokedactivation of human ASIC1a stably transfected in CHO cells. FIG. 3Adisplays the dose-dependent effect of Compound A and the benchmarkcompound amiloride on the size of the pH-evoked response. In thisexample, the size of the pH-evoked response was determined by measuringthe area under the curve of the response (total charge transfer) andnormalized to the control response. FIG. 3B depicts an example of anhASIC1a current (whole cell voltage clamp, −60 mV) evoked by a pH6.8-buffered solution (from pH 7.4) in the presence or absence ofCompound A (3 μM).

FIGS. 4A and 4B illustrate the inhibitory effects of Compound A onacid-induced activation of recombinant homomeric hASIC1a (FIG. 4A) andheteromeric hASIC1a+3 (FIG. 4B) channels, as described in Example 2.HEK293 cells were transfected either with hASIC1a alone orco-transfected with hASIC1a and hASIC3. Acid-induced inward currentswere recorded in the presence and absence of compound A (10 μM) usingthe whole-cell configuration of the patch-clamp method (voltage clampmode).

FIGS. 5A and 5B illustrate inhibitory effects of Compound A onacid-induced activation of recombinant homomeric hASIC1a (FIG. 5A) andheteromeric hASIC1a+3 (FIG. 5B) channels, as described in Example 3.Acid-induced currents were recorded from Xenopus laevis oocytes usingthe two-electrode voltage clamp method in the presence and absence ofCompound A (30 μM). Oocytes were microinjected with an hASIC1a encodingcDNA alone, or co-injected with hASIC1a and hASIC3 encoding cDNA. Thesedata show that Compound A effectively modulates the activity of thesegated ion channels.

FIGS. 6A and 6B illustrate the inhibitory effects of Compounds A (FIG.6A) and H (FIG. 6B) on native proton-gated currents recorded from ratdorsal root ganglion neurons in primary culture, as described in Example4. These endogenous proton-activated inward currents were recorded inthe presence and absence of Compound A (1 μM) or Compound H (1 μM) usingthe whole cell configuration of the patch clamp method (voltage clampmode). These data show that Compounds A and H can block native ASICresponses.

FIGS. 7A and 7B illustrate the inhibitory effects of Compounds A (FIG.7A) and H (FIG. 7B) on acid-induced action potential generation recordedfrom rat dorsal root ganglion neurons in primary culture, as describedin Example 4. The acid-evoked action potentials were recorded in thepresence and absence of Compound A (1 μM) or Compound H (1 μM) using thewhole cell configuration of the patch clamp method (current clamp mode).FIGS. 7A and 7B show that Compound A and H decrease the rate of theaction potential firing induced by pH 6.5 and 6.8, respectively.

FIG. 8 illustrates the effect of different concentrations of Compound Aon formalin-induced pain in rats. FIG. 8A depicts the total painbehavior (e.g., flinching, licking, biting) over time followingintraplantar injection of formalin and FIG. 8B displays the number oflicking and biting episodes. These results indicate that Compound Acauses a dose-dependent reduction of the pain behavior in the rat.

FIG. 9 depicts the dose-dependent effect of Compound A onFormalin-induced pain. FIG. 9A is the dose-response relationship ofCompound A on the total pain score (FIG. 8A) in phase IIa of theformalin test. The effective dose where the pain score is reduced byhalf (ED₅₀) is 12 mg/kg. FIG. 9B shows a linear relationship between thedose of Compound A and the plasma level 1.5 h after compoundadministration.

FIGS. 10A and 10B illustrate the effects of Compounds B and H,respectively, on spontaneous chemically-induced pain in the formalintest in rats, as described in Example 5. These results indicate thatboth compounds cause a dose-dependent reduction of the pain intensity asevaluated by the flinching behavior.

FIGS. 11A, 11B and 11C illustrate the effects of Compounds A, Compound Hand morphine, respectively, on inflammatory pain in rats, as describedin Example 6. Inflammation was induced by injecting complete Freund'sadjucant (CFA) in the hind paw. These results indicate that Compound Aand H cause a dose-dependent reduction of the pain intensity andnocifensive behavior as measured by the incapacitance meter (hindpawweight bearing difference).

FIG. 12 illustrates dose-dependent analgesic effects of Compound A inthe CFA model of chronic inflammatory pain: In vivo dose-dependentreduction of CFA-induced thermal hyperalgesia by Compound A compared tovehicle treated rats. In this model, relative high doses of thebenchmark compounds morphine (6 mg/kg) and indomethacin (30 mg/kg) werefully efficacious.

FIGS. 13A and 13B illustrate dose-response curves of the inhibitoryeffect of compounds J and K on hASIC1a activity as described inExample 1. HEK293 cells were transfected with hASIC1a and cells wereexposed to acidic buffer in the absence and presence of increasingconcentrations of Compounds J or K. Gated-channel activity wasdetermined by measuring intracellular calcium variation using acalcium-selective fluorescent dye. Compounds E, F and G dose-dependentlyinhibit acid-induced hASIC1A activity.

FIGS. 14A and 14B illustrate the inhibitory effects of Compounds J (FIG.14A) and K (FIG. 14B) on proton-gated currents recorded from CHO cellsexpressing hASIC1a in Example 2. These endogenous proton-activatedinward currents were recorded in the presence and absence of Compound J(1 μM) or Compound K (1 μM) using the whole cell configuration of thepatch clamp method (voltage clamp mode).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the identificationof compounds useful in modulation of the activity of gated ion channels.Gated ion channels are involved in receiving, conducting, andtransmitting signals in a cell (e.g., an electrically excitable cell,for example, a neuronal or muscle cell). Gated ion channels candetermine membrane excitability (the ability of, for example, a cell torespond to a stimulus and to convert it into a sensory impulse). Gatedion channels can also influence the resting potential of membranes, waveforms and frequencies of action potentials, and thresholds ofexcitation. Gated ion channels are typically expressed in electricallyexcitable cells, e.g., neuronal cells, and are multimeric; they can formhomomultimeric (e.g., composed of one type of subunit), orheteromultimeric structures (e.g., composed of more than one type ofsubunit). Gated ion channels can also be found in nonexcitable cells(e.g., adipose cells or liver cells), where they can play a role in, forexample, signal transduction.

Gated ion channels that are the focus of this invention are generallyhomomeric or heteromeric complexes composed of subunits, comprising atleast one subunit belonging to the DEG/ENaC, TRPV and/or P2X genesuperfamilies. Non-limiting examples of the DEG/ENaC receptor genesuperfamily include epithelial Na⁺ channels, e.g., αENaC, βENaC, γENaC,and/or δENaC, and the acid sensing ion channels (ASICs), e.g., ASIC1,ASIC1a, ASIC1b, ASIC2, ASIC2a, ASIC2b, ASIC3, and/or ASIC4. Non-limitingexamples of the P2X receptor gene superfamily include P2X₁, P2X₂, P2X₃,P2X₄, P2X₅, P2X₆, and P2X₇. Non-limiting examples of the TRPV receptorgene superfamily include TRPV1 (also referred to as VR1), TRPV2 (alsoreferred to as VRL-1), TRPV3 (also referred to as VRL-3), TRPV4 (alsoreferred to as VRL-2), TRPV5 (also referred to as ECAC-1), and/or TRPV6(also referred to as ECAC-2).

Non limiting examples of heteromultimeric gated ion channels includeαENaC, βENaC and γENaC; αENaC, βENaC and δENaC; ASIC1a and ASIC2a;ASIC1a and ASIC2b; ASIC1a and ASIC3; ASIC1b and ASIC3; ASIC2a andASIC2b; ASIC2a and ASIC3; ASIC2b and ASIC3; ASIC1a, ASIC2a and ASIC3;ASIC3 and P2X, e.g. P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆ and P2X₇,preferably ASIC3 and P2X₂; ASIC3 and P2X₃; and ASIC3, P2X₂ and P2X₃ASIC4 and at least one of ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3;BLINaC (or hINaC) and at least one of ASIC1a, ASIC1b, ASIC2a, ASIC2b,ASIC3, and ASIC4; δENaC and ASIC, e.g. ASIC1a, ASIC1b, ASIC2a, ASIC2b,ASIC3 and ASIC4; P2X₁ and P2X₂, P2X₁ and P2X₅, P2X₂ and P2X₃, P2X₂ andP2X₆, P2X₄ and P2X₆, TRPV1 and TRPV2, TRPV5 and TRPV6, TRPV1 and TRPV4.

Based on the above, there is a need for compositions which modulate theactivity of ion channels and methods of use thereof for the treatment ofconditions, diseases and disorders related to pain, inflammation, theneurological system, the gastrointestinal system and genitourinarysystem.

DEFINITIONS

As used herein, the term “acid” refers to carboxylic acid, sulfonicacid, sulfinic acid, sulfamic acid, phosphonic acid and boronic acidfunctional groups.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.Furthermore, the expression “C_(x)-C_(y)-alkyl”, wherein x is 1-5 and yis 2-10 indicates a particular alkyl group (straight- or branched-chain)of a particular range of carbons. For example, the expressionC₁-C₄-alkyl includes, but is not limited to, methyl, ethyl, propyl,butyl, isopropyl, tert-butyl and isobutyl.

The term alkyl further includes alkyl groups which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In an embodiment, a straight chainor branched chain alkyl has 10 or fewer carbon atoms in its backbone(e.g., C₁-C₁₀ for straight chain, C₃-C₁₀ for branched chain), and morepreferably 6 or fewer carbons. Likewise, preferred cycloalkyls have from4-7 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure.

Moreover, alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl,etc.) includes both “unsubstituted alkyl” and “substituted alkyl”, thelatter of which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone, which allowthe molecule to perform its intended function. The term “substituted” isintended to describe moieties having substituents replacing a hydrogenon one or more atoms, e.g. C, O or N, of a molecule. Such substituentscan include, for example, alkenyl, alkynyl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclic, alkylaryl, morpholino, phenol, benzyl, phenyl, piperizine,cyclopentane, cyclohexane, pyridine, 5H-tetrazole, triazole, piperidine,or an aromatic or heteroaromatic moiety.

Further examples of substituents of the invention, which are notintended to be limiting, include moieties selected from straight orbranched alkyl (preferably C₁-C₅), cycloalkyl (preferably C₃-C₈), alkoxy(preferably C₁-C₆), thioalkyl (preferably C₁-C₆), alkenyl (preferablyC₂-C₆), alkynyl (preferably C₂-C₆), heterocyclic, carbocyclic, aryl(e.g., phenyl), aryloxy (e.g., phenoxy), aralkyl (e.g., benzyl),aryloxyalkyl (e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl,heteroaralkyl, alkylcarbonyl and arylcarbonyl or other such acyl group,heteroarylcarbonyl, or heteroaryl group, (CR′R″)₀₋₃NR′R″ (e.g., —NH₂),(CR′R″)₀₋₃CN (e.g., —CN), —NO₂, halogen (e.g., —F, —Cl, —Br, or —I),(CR′R″)₀₋₃C(halogen)₃ (e.g., —CF₃), (CR′R″)₀₋₃CH(halogen)₂,(CR′R″)₀₋₃CH₂(halogen), (CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃(CNH)NR′R″,(CR′R″)₀₋₃S(O)₁₋₂NR′R″, (CR′R″)₀₋₃CHO, (CR′R″)₀₋₃O(CR′R″)₀₋₃H,(CR′R″)₀₋₃S(O)₀₋₃R′ (e.g., —SO₃H, —OSO₃H), (CR′R″)₀₋₃O(CR′R″)₀₋₃H (e.g.,—CH₂OCH₃ and —OCH₃), (CR′R″)₀₋₃S(CR′R″)₀₋₃H (e.g., —SH and —SCH₃),(CR′R″)₀₋₃OH (e.g., —OH), (CR′R″)₀₋₃COR′, (CR′R″)₀₋₃ (substituted orunsubstituted phenyl), (CR′R″)₀₋₃(C₃-C₈ cycloalkyl), (CR′R″)₀₋₃CO₂R′(e.g., —CO₂H), or (CR′R″)₀₋₃OR′ group, or the side chain of anynaturally occurring amino acid; wherein R′ and R″ are each independentlyhydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, or aryl group.Such substituents can include, for example, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, oxime, thiol, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, or an aromatic or heteroaromatic moiety. In certainembodiments, a carbonyl moiety (C═O) can be further derivatized with anoxime moiety, e.g., an aldehyde moiety can be derivatized as its oxime(—C═N—OH) analog. It will be understood by those skilled in the art thatthe moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. Cycloalkyls can be further substituted,e.g., with the substituents described above. An “aralkyl” moiety is analkyl substituted with an aryl (e.g., phenylmethyl (i.e., benzyl)).

The term “amine” or “amino” should be understood as being broadlyapplied to both a molecule, or a moiety or functional group, asgenerally understood in the art, and can be primary, secondary, ortertiary. The term “amine” or “amino” includes compounds where anitrogen atom is covalently bonded to at least one carbon, hydrogen orheteroatom. The terms include, for example, but are not limited to,“alkyl amino,” “arylamino,” “diarylamino,” “alkylarylamino,”“alkylaminoaryl,” “arylaminoalkyl,” “alkaminoalkyl,” “amide,” “amido,”and “aminocarbonyl.” The term “alkyl amino” comprises groups andcompounds wherein the nitrogen is bound to at least one additional alkylgroup. The term “dialkyl amino” includes groups wherein the nitrogenatom is bound to at least two additional alkyl groups. The term“arylamino” and “diarylamino” include groups wherein the nitrogen isbound to at least one or two aryl groups, respectively. The term“alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl” refers to anamino group which is bound to at least one alkyl group and at least onearyl group. The term “alkaminoalkyl” refers to an alkyl, alkenyl, oralkynyl group bound to a nitrogen atom which is also bound to an alkylgroup.

The term “amide,” “amido” or “aminocarbonyl” includes compounds ormoieties which contain a nitrogen atom which is bound to the carbon of acarbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl”or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl and arylcarbonylamino groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,”“arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,”“alkynylcarbonylamino,” and “arylcarbonylamino” are included in term“amide.” Amides also include urea groups (aminocarbonylamino) andcarbamates (oxycarbonylamino).

In a particular embodiment of the invention, the term “amine” or “amino”refers to substituents of the formulas N(R⁸)R⁹ or C₁₋₆—N(R⁸)R⁹, whereinR⁸ and R⁹ are each, independently, selected from the group consisting of—H and —(C₁₋₄alkyl)₀₋₁G, wherein G is selected from the group consistingof —COOH, —H, —PO₃H, —SO₃H, —Br, —Cl, —F, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl,aryl, —C(O)OC₁-C₆-alkyl, —C(O)C₁₋₄alkyl-COOH, —C(O)C₁-C₄-alkyl and—C(O)-aryl; or N(R⁸)R⁹ is pyrrolyl, tetrazolyl, pyrrolidinyl,pyrrolidinyl-2-one, dimethylpyrrolyl, imidazolyl and morpholino.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that can include from zero to four heteroatoms, forexample, phenyl, pyrrole, furan, thiophene, thiazole, isothiaozole,imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine,pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, theterm “aryl” includes multicyclic aryl groups, e.g., tricyclic, bicyclic,e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole,benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,isoquinoline, anthryl, phenanthryl, napthridine, indole, benzofuran,purine, benzofuran, deazapurine, or indolizine. Those aryl groups havingheteroatoms in the ring structure can also be referred to as “arylheterocycles”, “heterocycles,” “heteroaryls” or “heteroaromatics.” Thearomatic ring can be substituted at one or more ring positions with suchsubstituents as described above, as for example, alkyl, halogen,hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino(including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclic ringswhich are not aromatic so as to form a polycycle (e.g., tetralin).

It will be noted that the structures of some of the compounds of thisinvention include asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention. Such isomers can be obtained in substantially pure form byclassical separation techniques and by stereochemically controlledsynthesis. Furthermore, the structures and other compounds and moietiesdiscussed in this application also include all tautomers thereof.Compounds described herein can be obtained through art recognizedsynthesis strategies.

Additionally, the phrase “any combination thereof” implies that anynumber of the listed functional groups and molecules can be combined tocreate a larger molecular architecture. For example, the terms “phenyl,”“carbonyl” (or “═O”), “—O—,” “—OH,” and C₁₋₆ (i.e., —CH₃ and—CH₂CH₂CH₂—) can be combined to form a 3-methoxy-4-propoxybenzoic acidsubstituent. It is to be understood that when combining functionalgroups and molecules to create a larger molecular architecture,hydrogens can be removed or added, as required to satisfy the valence ofeach atom.

As used herein, the terms “gated ion channel” or “gated channel” areused interchangeably and are intended to refer to a mammalian (e.g.,rat, mouse, human) multimeric complex responsive to, for example,variations of voltage (e.g., membrane depolarization orhyperpolarization), temperature (e.g., higher or lower than 37° C.), pH(e.g., pH values higher or lower than 7.4), ligand concentration and/ormechanical stimulation. Examples of specific modulators include, but arenot limited to, endogenous extracellular ligands such as anandamide,ATP, glutamate, cysteine, glycine, gamma-aminobutyric acid (GABA),histidine, serotonin (5HT), acetylcholine, epinephrine, norepinephrine,protons, ions, e.g., Na⁺, Ca⁺⁺, K⁺, Zn⁺, and/or peptides, e.g.,Met-enkephaline, Leu-enkephaline, dynorphin, neurotrophins, and/or theRFamide related peptides, e.g., FMRFamide and/or FLRFamide; toendogenous intracellular ligands such as cyclic nucleotides (e.g.cyclicAMP, cyclicGMP), ATP, Ca⁺⁺ and/or G-proteins; to exogenousextracellular ligands or modulators such asα-amino-3-hydroxy-5-methyl-4-isolaxone propionate (AMPA), amiloride,capsaicin, capsazepine, epibatidine, cadmium, barium, gadolinium,guanidium, kainate, N-methyl-D-aspartate (NMDA). Gated ion channels alsoinclude complexes responsive to toxins, examples of which include, butare not limited to, Agatoxin (e.g. α-agatoxin. IVA, IVB, ω-agatoxin IVA,TK), Agitoxins (Agitoxin 2), Apamin, Argiotoxins, Batrachotoxins,Brevetoxins (e.g. Brevctoxin PbTx-2, PbTx-3, PbTx-9), Charybdotoxins,Chlorotoxins, Ciguatoxins, Conotoxins (e.g. a-conotoxin GI, GIA, GII,IMI, MI, MII, SI, SIA, SII, and/or EI; δ-conotoxins, μ-conotoxin GIIIA,GIIIB, GIIIC and/or GS, ω-conotoxin GVIA, MVIIA MVIIC, MVIID, SVIAand/or SVIB), Dendrotoxins, Grammotoxins (GsMTx-4, ω-grammotoxin SIA),Grayanotoxins, Hanatoxins, Iberiotoxins, Imperatoxins, Jorotoxins,Kaliotoxins, Kurtoxins, Leiurotoxin 1, Pricotoxins, Psalmotoxins, (e.g.,Psalmotoxin 1 (PcTx1)), Margatoxins, Noxiustoxins, Phrixotoxins, PLTXII, Saxitoxins, Stichodactyla Toxins, sea anemone toxins (e.g. APETx2from Anthopleura elegantissima), Tetrodotoxins, Tityus toxin K-α,Scyllatoxins and/or tubocurarine.

In a preferred embodiment, the compounds of the invention modulate theactivity of ASIC1a and/or ASIC3.

“Gated ion channel-mediated activity” is a biological activity that isnormally modulated (e.g., inhibited or promoted), either directly orindirectly, in the presence of a gated ion channel. Gated ionchannel-mediated activities include, for example, receiving,integrating, transducing, conducting, and transmitting signals in acell, e.g., a neuronal or muscle cell. A biological activity that ismediated by a particular gated ion channel, e.g. ASIC la or ASIC3, isreferred to herein by reference to that gated ion channel, e.g. AS IC la- or ASIC3-mediated activity. To determine the ability of a compound toinhibit a gated ion channel-mediated activity, conventional in vitro andin vivo assays can be used which are described herein.

“Neurotransmission,” as used herein, is a process by which smallsignaling molecules, termed neurotransmitters, are rapidly passed in aregulated fashion from a neuron to another cell. Typically, followingdepolarization associated with an incoming action potential, aneurotransmitter is secreted from the presynaptic neuronal terminal. Theneurotransmitter then diffuses across the synaptic cleft to act onspecific receptors on the postsynaptic cell, which is most often aneuron but can also be another cell type (such as muscle fibers at theneuromuscular junction). The action of neurotransmitters can either beexcitatory, depolarizing the postsynaptic cell, or inhibitory, resultingin hyperpolarization. Neurotransmission can be rapidly increased ordecreased by neuromodulators, which typically act eitherpre-synaptically or post-synaptically. The gated ion channel ASIC1a hasbeen shown to possibly contribute to neurotransmission [Babini et al., JBiol. Chem. 277(44):41597-603 (2002)].

Examples of gated ion channel-mediated activities include, but are notlimited to, pain (e.g., inflammatory pain, acute pain, chronic malignantpain, chronic nonmalignant pain and neuropathic pain), inflammatorydisorders, diseases and disorders of the genitourinary andgastrointestinal systems, and neurological disorders (e.g.,neurodegenerative or neuropsychiatric disorders).

“Pain” is defined as an unpleasant sensory and emotional experienceassociated with actual or potential tissue damage, or described in termsof such damage (International Association for the Study of Pain—IASP).Pain is classified most often based on duration (i.e., acute vs. chronicpain) and the underlying pathophysiology (i.e., nociceptive vs.neuropathic pain).

Acute pain can be described as an unpleasant experience with emotionaland cognitive, as well as sensory, features that occur in response totissue trauma and disease and serves as a defensive mechanism. Acutepain is usually accompanied by a pathology (e.g., trauma, surgery,labor, medical procedures, acute disease states) and the pain resolveswith healing of the underlying injury. Acute pain is mainly nociceptive,but can also be neuropathic.

Chronic pain is pain that extends beyond the period of healing, withlevels of identified pathology that often are low and insufficient toexplain the presence, intensity and/or extent of the pain (American PainSociety—APS). Unlike acute pain, chronic pain serves no adaptivepurpose. Chronic pain can be nociceptive, neuropathic, or both andcaused by injury (e.g., trauma or surgery), malignant conditions, or avariety of chronic conditions (e.g., arthritis, fibromyalgia andneuropathy). In some cases, chronic pain exists de novo with no apparentcause.

“Nociceptive pain” is pain that results from damage to tissues andorgans. Nociceptive pain is caused by the ongoing activation of painreceptors in either the superficial or deep tissues of the body.Nociceptive pain is further characterized as “somatic pain”, including“cutaneous pain” and “deep somatic pain”, and “visceral pain”.

“Somatic pain” includes “cutaneous pain” and “deep somatic pain.”Cutaneous pain is caused by injury, diseases and disorders of the skinand related organs. Examples of conditions associated with cutaneouspain include, but are not limited to, cuts, burns, infections,lacerations, as well as traumatic injury and post-operative or surgicalpain (e.g., at the site of incision).

“Deep somatic pain” results from injuries, diseases or disorders of themusculoskeletal tissues, including ligaments, tendons, bones, bloodvessels and connective tissues. Examples of deep somatic pain orconditions associated with deep somatic pain include, but are notlimited to, sprains, broken bones, arthralgia, vasculitis, myalgia andmyofascial pain. Arthralgia refers to pain caused by a joint that hasbeen injured (such as a contusion, break or dislocation) and/or inflamed(e.g., arthritis). Vaculitis refers to inflammation of blood vesselswith pain. Myalgia refers to pain originating from the muscles.Myofascial pain refers to pain stemming from injury or inflammation ofthe fascia and/or muscles.

“Visceral” pain is associated with injury, inflammation or disease ofthe body organs and internal cavities, including but not limited to, thecirculatory system, respiratory system, gastrointestinal system,genitourinary system, immune system, as well as ear, nose and throat.Visceral pain can also be associated with infectious and parasiticdiseases that affect the body organs and tissues. Visceral pain isextremely difficult to localize, and several injuries to visceral tissueexhibit “referred” pain, where the sensation is localized to an areacompletely unrelated to the site of injury. For example, myocardialischaemia (the loss of blood flow to a part of the heart muscle tissue)is possibly the best known example of referred pain; the sensation canoccur in the upper chest as a restricted feeling, or as an ache in theleft shoulder, arm or even hand. Phantom limb pain is the sensation ofpain from a limb that one no longer has or no longer gets physicalsignals from—an experience almost universally reported by amputees andquadriplegics.

“Neuropathic pain” or “neurogenic pain” is pain initiated or caused by aprimary lesion, dysfunction or perturbation in the nervous system.“Neuropathic pain” can occur as a result of trauma, inflammation ordisease of the peripheral nervous system (“peripheral neuropathic pain”)and the central nervous system (“central pain”). For example,neuropathic pain can be caused by a nerve or nerves that are irritated,trapped, pinched, severed or inflamed (neuritis). There are manyneuropathic pain syndromes, such as diabetic neuropathy, trigeminalneuralgia, postherpetic neuralgia (“shingles”), post-stroke pain, andcomplex regional pain syndromes (also called reflex sympatheticdystrophy or “RSD” and causalgia).

As used herein, the term “inflammatory disease or disorder” includesdiseases or disorders which are caused, at least in part, or exacerbatedby, inflammation, which is generally characterized by increased bloodflow, edema, activation of immune cells (e.g., proliferation, cytokineproduction, or enhanced phagocytosis), heat, redness, swelling, pain andloss of function in the affected tissue and organ. The cause ofinflammation can be due to physical damage, chemical substances,micro-organisms, tissue necrosis, cancer or other agents. Inflammatorydisorders include acute inflammatory disorders, chronic inflammatorydisorders, and recurrent inflammatory disorders. Acute inflammatorydisorders are generally of relatively short duration, and last for fromabout a few minutes to about one to two days, although they can lastseveral weeks. The main characteristics of acute inflammatory disordersinclude increased blood flow, exudation of fluid and plasma proteins(edema) and emigration of leukocytes, such as neutrophils. Chronicinflammatory disorders, generally, are of longer duration, e.g., weeksto months to years or longer, and are associated histologically with thepresence of lymphocytes and macrophages and with proliferation of bloodvessels and connective tissue. Recurrent inflammatory disorders includedisorders which recur after a period of time or which have periodicepisodes. Some disorders can fall within one or more categories.

The terms “neurological disorder” and “neurodegenerative disorder” referto injuries, diseases and dysfunctions of the nervous system, includingthe peripheral nervous system and central nervous system. Neurologicaldisorders and neurodegenerative disorders include, but are not limitedto, diseases and disorders that are associated with gated ionchannel-mediated biological activity. Examples of neurological disordersinclude, but are not limited to, Alzheimer's disease, epilepsy, cancer,neuromuscular diseases, multiple sclerosis, amyotrophic lateralsclerosis, stroke, cerebral ischemia, neuropathy (e.g.,chemotherapy-induced neuropathy, diabetic neuropathy), retinal pigmentdegeneration, Huntington's chorea, and Parkinson's disease, anxietydisorders (e.g., phobic disorders (e.g., agoraphobia, claustrophobia),panic disorders, phobias, anxiety hysteria, generalized anxietydisorder, and neurosis), and ataxia-telangiectasia.

As used herein, “neuropathy” is defined as a failure of the nerves thatcarry information to and from the brain and spinal cord resulting in oneor more of pain, loss of sensation, and inability to control muscles. Insome cases, the failure of nerves that control blood vessels,intestines, and other organs results in abnormal blood pressure,digestion problems, and loss of other basic body processes. Peripheralneuropathy can involve damage to a single nerve or nerve group(mononeuropathy) or can affect multiple nerves (polyneuropathy).

The term “treated,” “treating” or “treatment” includes the diminishmentor alleviation of at least one symptom associated with the pain,inflammatory disorder, neurological disorder, genitourinary disorder orgastrointestinal disorder (e.g., a symptom associated with or caused bygated ion channel mediated activity) being treated. In certainembodiments, the treatment comprises the modulation of the interactionof a gated ion channel (e.g., ASIC1a and/or ASIC3) by a gated ionchannel modulating compound, which would in turn diminish or alleviateat least one symptom associated with or caused by the gated ionchannel-mediated activity being treated. For example, treatment can bediminishment of one or several symptoms of a disorder or completeeradication of a disorder.

As used herein, the phrase “therapeutically effective amount” of thecompound is the amount necessary or sufficient to treat or prevent pain,an inflammatory disorder, a neurological disorder, a gastrointestinaldisorder or a genitourinary disorder, (e.g., to prevent the varioussymptoms of a gated ion channel-mediated activity). In an example, aneffective amount of the compound is the amount sufficient to alleviateat least one symptom of the disorder, e.g., pain, inflammation, aneurological disorder, a gastrointestinal disorder or a genitourinarydisorder, in a subject.

The term “subject” is intended to include animals, which are capable ofsuffering from or afflicted with a gated ion channel-associated state orgated ion channel-associated disorder, or any disorder involving,directly or indirectly, gated ion channel activity. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In certainembodiments, the subject is a human, e.g., a human suffering from, atrisk of suffering from, or potentially capable of suffering from pain,inflammation, a neurological disorder, a gastrointestinal disorder or agenitourinary disorder (e.g. associated with gated channel-associatedactivity).

The language “gated ion channel modulator” refers to compounds thatmodulate, i.e., inhibit, promote or otherwise alter the activity of agated ion channel. For example, the gated ion channel modulator caninhibit, promote or otherwise alter the response of a gated ion channelto, for example, variations of voltage (e.g., membrane depolarization orhyperpolarization), temperature (e.g., higher or lower than 37° C.), pH(e.g., pH values higher or lower than 7.4), ligand concentration and/ormechanical stimulation. Examples of gated ion channel modulators includecompounds of the invention (i.e., Formulas 1, 2, 3, 4 and 5, as well ascompounds A, B, C, D, E, F, G, H, I, J and K) including salts thereof,e.g., a pharmaceutically acceptable salt. In a particular embodiment,the gated ion channel modulators of the invention can be used to treat adisease or disorder associated with pain, inflammation, neurologicaldisorders, gastrointestinal disorders or genitourinary disorders in asubject in need thereof. In another embodiment, the compounds of theinvention can be used to treat an inflammatory disorder in a subject inneed thereof.

Modulators of Ion Channel Activity

The present invention provides compounds which modulate the activity ofa gated ion channel. In some embodiments, the compounds of the inventionmodulate the activity of a gated ion channel comprised of at least onesubunit belonging to the DEG/ENaC, TRPV and/or P2X gene superfamilies.In some embodiments, the compounds of the invention modulate theactivity of the gated ion channel comprised of at least one subunitselected from the group consisting of αENaC, βENaC, γENaC, δENaC,ASIC1a, ASIC 1b, ASIC2a, ASIC2b, ASIC3, ASIC4, BLINaC, hINaC, P2X₁,P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, P2X₇, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5,and TRPV6. In still other embodiments, the compounds of the inventionmodulate the activity of the DEG/ENaC gated ion channel comprised of atleast one subunit selected from the group consisting of αENaC, βENaC,γENaC, δENaC, BLINaC, hINaC, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, andASIC4. In certain embodiments, the compounds of the invention modulatethe activity of the DEG/ENaC gated ion channel comprised of at least onesubunit selected from the group consisting of ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3, and ASIC4. In certain embodiments, the compounds of theinvention modulate the activity of the DEG/ENaC gated ion channelcomprised of at least two subunits selected from the group consisting ofASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. In yet otherembodiments, the compounds of the invention modulate the activity of theDEG/ENaC gated ion channel comprised of at least three subunits selectedfrom the group consisting of ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, andASIC4. In certain embodiments, the compounds of the invention modulatethe activity of a gated ion channel comprised of ASIC, i.e., ASIC1a orASIC1b. In certain embodiments, the compounds of the invention modulatethe activity of a gated ion channel comprised of ASIC3. In certainembodiments, the compounds of the invention modulate the activity of agated ion channel comprised of ASIC1a and ASIC2a; ASIC1a and ASIC3;ASIC1b and ASIC3; ASIC2a and ASIC2b; ASIC2a and ASIC3; ASIC2b and ASIC3;and ASIC1a, ASIC2a and ASIC3. In other embodiments, the compounds of theinvention modulate the activity of the P2X gated ion channel comprisedof at least one subunit selected from the group consisting of P2X₁,P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, and P2X₇. In certain embodiments, thecompounds of the invention modulate the activity of a gated ion channelcomprised of P2X₂, P2X₃ or P2X₄. In certain embodiments, the compoundsof the invention modulate the activity of a gated ion channel comprisedof P2X₁ and P2X₂, P2X₁ and P2X₅, P2X₂ and P2X₃, P2X₂ and P2X₆, and P2X₄and P2X₆. In yet another aspect of the invention, the compounds of theinvention modulate the activity of the TRPV gated ion channel comprisedof at least one subunit selected from the group TRPV1, TRPV2, TRPV3,TRPV4, TRPV5, and TRPV6. In certain embodiments, the compounds of theinvention modulate the activity of a gated ion channel comprised ofTRPV1 or TRPV2. In certain embodiments, the compounds of the inventionmodulate the activity of a gated ion channel comprised of TRPV I andTRPV2, TRPV1 and TRPV4, and TRPV5 and TRPV6.

In a particular embodiment, the compounds of the invention modulate theactivity of ASIC1a and/or ASIC3.

The nomenclature of the compounds of the present invention followsstandard conventions. The “southern” phenyl ring is numbered asindicated below:

In one aspect, the compound that modulates the activity of a gated ionchannel is of the Formula 1,

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof;

wherein the dashed lines indicate a single or double bond; R¹ isselected from the group consisting of hydrogen, alkyl, alkoxy-alkyl,hydroxy-alkyl, alkoxy-carbonyl-alkyl, alkyl-carbonyl-oxy-alkyl,cycloalkyl, cycloalkyl-alkyl, alkenyl, alkynyl, alkoxy, sulfonamide,amino, sulfonyl, sulfonic acid, urea, phenyl or benzyl, in which thephenyl or benzyl group is optionally substituted with halogen, CF₃,nitro, amino, cyano, hydroxy-alkyl, alkoxy, sulfonamide, alkenyl,alkynyl, amino, sulfonyl, sulfonic acid and urea; R² is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,—(CH₂)₁₋₄S(O)₃H, —C(O)C₁₋₄alkyl and S(O)₂C₁₋₄alkyl; R³ is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, acyl, phenyl, benzyl,—COOH, —C(O)N(CH₃)₂, —O-phenyl, —OCF₃, alkoxy, —O(CH₂)₀₋₄OCH₃, —C(O)H,—C(O)CH₃,

and R⁴ and R⁵ are each, independently, selected from the groupconsisting of halogen, CF₃, nitro, amino, cyano, hydroxyl, alkyl,alkoxy, phenoxy and phenyl, or a group of the formula —SO₂NR′R″, whereinR′ and R″ independently of each another represents hydrogen or alkyl.

In one embodiment of Formula 1, R¹ is selected from the group consistingof hydrogen, alkyl, alkenyl, and alkynyl; R² is selected from the groupconsisting of hydrogen, hydroxyl, alkyl, alkenyl and alkynyl; R³ isselected from the group consisting of hydrogen, hydroxyl, alkyl, acyl,phenyl, benzyl, and COOH; and R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, CF₃, nitro, amino, cyano,hydroxyl, alkyl, alkoxy, phenoxy and phenyl.

In another embodiment of Formula 1, R¹ is selected from the groupconsisting of hydrogen and alkyl; R² is selected from the groupconsisting of hydrogen, hydroxyl, alkyl, alkenyl and alkynyl; R³ isselected from the group consisting of hydrogen, hydroxyl, alkoxy andalkyl; and R⁴ and R⁵ are each, independently, selected from the groupconsisting of halogen, CF₃, alkyl, phenoxy and alkoxy.

In yet another embodiment of Formula 1, the dashed lines indicate adouble bond; R¹ is selected from the group consisting of alkyl; R² isselected from the group consisting of hydrogen and alkyl; R³ ishydroxyl; and R⁴ and R⁵ are each, independently, selected from the groupconsisting of halogen, CF₃, alkyl, phenoxy and alkoxy.

A preferred embodiment of Formula 1 is represented as Formula 2,

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof;

wherein R¹ is selected from the group consisting of hydrogen, alkyl,alkoxy-alkyl, alkoxy-carbonyl-alkyl, alkyl-carbonyl-oxy-alkyl,cycloalkyl, cycloalkyl-alkyl, alkenyl, alkynyl, alkoxy, sulfonamide,amino, sulfonyl, sulfonic acid, urea, phenyl or benzyl, in which thephenyl or benzyl group is optionally substituted with halogen, CF₃,nitro, amino, cyano, hydroxy-alkyl, alkoxy, sulfonamide, alkenyl,alkynyl, amino, sulfonyl, sulfonic acid and urea; R² is selected fromthe group consisting of hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,—(CH₂)₁₋₄S(O)₃H, —C(O)C₁₋₄alkyl and S(O)₂C₁₋₄alkyl; and R⁴ and R⁵ areeach, independently, selected from the group consisting of halogen, CF₃,nitro, amino, cyano, hydroxyl, alkyl, alkoxy, phenoxy and phenyl, or agroup of the formula —SO₂NR′R″, wherein R′ and R″ independently of eachanother represents hydrogen or alkyl.

In one embodiment of Formula 2, R¹ is selected from the group consistingof hydrogen, alkyl, alkenyl, and alkynyl; R² is selected from the groupconsisting of hydrogen, hydroxyl, alkyl, alkenyl and alkynyl; and R⁴ andR⁵ are each, independently, selected from the group consisting ofhalogen, CF₃, nitro, amino, cyano, hydroxyl, alkyl, alkoxy, phenoxy andphenyl.

In yet another embodiment of Formula 2, R¹ is selected from the groupconsisting of hydrogen and alkyl; R² is selected from the groupconsisting of hydrogen, hydroxyl, alkyl, alkenyl and alkynyl; and R⁴ andR⁵ are each, independently, selected from the group consisting ofhalogen, CF₃, alkyl, phenoxy and alkoxy.

In still another embodiment of Formula 2, R¹ is selected from the groupconsisting of alkyl; R² is selected from the group consisting ofhydrogen and alkyl; and R⁴ and R⁵ are each, independently, selected fromthe group consisting of halogen, CF₃, alkyl, phenoxy and alkoxy.

In one embodiment of Formula 2, R¹ is selected from the group consistingof hydrogen, C₁₋₄-alkyl, C₁₋₄-alkenyl, and C₁₋₄-alkynyl; R² is selectedfrom the group consisting of hydrogen, hydroxyl, C₁₋₄-alkyl,C₁₋₄-alkenyl and C₁₋₄-alkynyl; and R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, CF₃, nitro, amino, cyano,hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkoxy, phenoxy and phenyl.

In yet another embodiment of Formula 2, R¹ is selected from the groupconsisting of hydrogen and C₁₋₄-alkyl; R² is selected from the groupconsisting of hydrogen, hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkenyl andC₁₋₄-alkynyl; and R⁴ and R⁵ are each, independently, selected from thegroup consisting of halogen, CF₃, C₁₋₄-alkyl, phenoxy and C₁₋₄-alkoxy.

In still another embodiment of Formula 2, R¹ is selected from the groupconsisting of C₁₋₄-alkyl; R² is selected from the group consisting ofhydrogen and C₁₋₄-alkyl; and R⁴ and R⁵ are each, independently, selectedfrom the group consisting of halogen, CF₃, C₁₋₄-alkyl, phenoxy andC₁₋₄-alkoxy.

In another embodiment of Formula 2, R¹ is selected from the groupconsisting of —CH₃ and —CH₂CH₃; R² is selected from the group consistingof hydrogen; and R⁴ and R⁵ are each, independently, selected from thegroup consisting of halogen, alkyl and alkoxy.

In another embodiment of Formula 2, R¹ is selected from the groupconsisting of —CH₃ and —CH₂CH₃; R² is selected from the group consistingof hydrogen; and R⁴ and R⁵ are each, independently, selected from thegroup consisting of halogen, C₁₋₄-alkyl and C₁₋₄-alkoxy.

In another embodiment of Formula 2, R¹ is CH₃ or CH₂CH₃.

In yet another embodiment of Formula 2, R² is H.

In still another embodiment of Formula 2, R⁴ is halogen.

In another embodiment of Formula 2, R⁵ is alkoxy.

In yet another embodiment of Formula 2, R⁴ is fluoro or chloro.

In another embodiment of Formula 2, R⁵ is OCH₃.

In still another embodiment of Formula 2, R⁴ and R⁵ are both alkyl.

In another embodiment of Formula 2, R⁴ and R⁵ are both CH₃.

Another preferred embodiment of Formula 1 is represented as Formula 3,

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof;

wherein R¹ is selected from the group consisting of hydrogen, alkyl,alkoxy-alkyl, alkoxy-carbonyl-alkyl, alkyl-carbonyl-oxy-alkyl,cycloalkyl, cycloalkyl-alkyl, alkenyl, alkynyl, alkoxy, sulfonamide,amino, sulfonyl, sulfonic acid, urea phenyl or benzyl, in which thephenyl or benzyl group is optionally substituted with halogen, CF₃,nitro, amino, cyano, hydroxy-alkyl, alkoxy, sulfonamide, alkenyl,alkynyl, amino, sulfonyl, sulfonic acid and urea; and R⁴ and R⁵ areeach, independently, selected from the group consisting of halogen,phenoxy, CF₃, nitro, amino, cyano, hydroxyl, alkyl, alkoxy and phenyl,or a group of the formula —SO₂NR′R″, wherein R′ and R″ independently ofeach another represents hydrogen or alkyl.

In a preferred embodiment of Formula 3, R⁵ is in the 2 position of thearyl ring and R⁴ is in the 5 position of the aryl ring, or R⁴ is in the3 position of the aryl ring and R⁵ is in the 5 position of the arylring.

In one embodiment of Formula 3, R¹ is selected from the group consistingof hydrogen, alkyl, alkenyl, and alkynyl; and R⁴ and R⁵ are each,independently, selected from the group consisting of halogen, CF₃,nitro, amino, cyano, hydroxyl, alkyl, alkoxy, phenoxy and phenyl.

In another embodiment of Formula 3, R¹ is selected from the groupconsisting of hydrogen and alkyl; and R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, CF₃, alkyl, phenoxy andalkoxy.

In yet another embodiment of Formula 3, R¹ is selected from the groupconsisting of alkyl; and R⁴ and R⁵ are each, independently, selectedfrom the group consisting of halogen, CF₃, alkyl, phenoxy and alkoxy.

In still another embodiment of Formula 3, R¹ is selected from the groupconsisting of —CH₃ and —CH₂CH₃; and R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, phenoxy and alkoxy.

In one embodiment of Formula 3, R¹ is selected from the group consistingof hydrogen, C₁₋₄-alkyl, C₁₋₄-alkenyl, and C₁₋₄-alkynyl; and R⁴ and R⁵are each, independently, selected from the group consisting of halogen,CF₃, nitro, amino, cyano, hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkoxy, phenoxy andphenyl.

In another embodiment of Formula 3, R¹ is selected from the groupconsisting of hydrogen and C₁₋₄-alkyl; and R⁴ and R⁵ are each,independently, selected from the group consisting of halogen, CF₃, Cphenoxy and C₁₋₄-alkoxy.

In yet another embodiment of Formula 3, R¹ is selected from the groupconsisting of C₁₋₄-alkyl; and R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, CF₃, C₁₋₄-alkyl, phenoxyand C₁₋₄-alkoxy.

In still another embodiment of Formula 3, R¹ is selected from the groupconsisting of —CH₃ and —CH₂CF₁₃; and R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, phenoxy and C₁₋₄-alkoxy.

In another embodiment of Formula 3, R¹ is CH₃ or CH₂CH₃.

In still another embodiment of Formula 3, R⁴ is halogen.

In yet another embodiment of Formula 3, R⁵ is alkoxy.

In another embodiment of Formula 3, R⁴ is fluoro or chloro.

In another embodiment of Formula 3, R⁵ is OCH₃.

In still another embodiment of Formula 3, R⁴ and R⁵ are both alkyl.

In another embodiment of Formula 4, R⁴ and R⁵ are both CH₃.

Another preferred embodiment of Formula 1 is represented as Formula 4,

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof;

wherein R¹ is selected from the group consisting of —CH₃ and —CH₂CH₃;and R⁴ and R⁵ are each, independently, selected from the groupconsisting of halogen, CF₃, alkyl, and alkoxy.

In a preferred embodiment of Formula 4, R⁵ is in the 2 position of thearyl ring and R⁴ is in the 5 position of the aryl ring, or R⁴ is in the3 position of the aryl ring and R⁵ is in the 5 position of the arylring.

In another preferred embodiment of Formula 4, R¹ is selected from thegroup consisting of —CH₃ and —CH₂CH₃; and R⁴ and R⁵ are each,independently, selected from the group consisting of halogen, CF₃,C₁₋₄-alkyl, and C₁₋₄-alkoxy.

In one embodiment of Formula 4, R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, alkyl and alkoxy. Inanother embodiment of Formula 4, R⁴ and R⁵ are each, independently,selected from the group consisting of halogen, C₁₋₄-alkyl andC₁₋₄-alkoxy.

In another embodiment of Formula 4, R⁴ is fluoro, and R⁵ is —OCH₃, R⁴ ischloro, and R⁵ is —OCH₃, or R⁴ and R⁵ are both CH₃.

A preferred embodiment of Formula 4 is5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound A):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound B):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound C):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(3,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound D):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(3,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound E):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(2,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound F):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound G):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 4 is5-(2,3-dimethyl-phenyl)-8-ethyl-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime (Compound I):

and pharmaceutically acceptable salts thereof.

In another aspect, the compound of the invention is of the Formula 5,

and pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof;

wherein R¹ is selected from the group consisting of hydrogen,C₁₋₄-alkyl, C₁₋₄-alkoxy-C₁₋₄-alkyl, C₁₋₄-alkoxy-carbonyl-C₁₋₄-alkyl,C₁₋₄-alkyl-carbonyl-oxy-C₁₋₄-alkyl, C₃₋₆-cycloalkyl,C₃₋₆-cycloalkyl-C₁₋₁₄-alkyl, C₁₋₄-alkenyl, C₁₋₄-alkynyl, C₁₋₄-alkoxy,sulfonamide, amino, sulfonyl, sulfonic acid, urea, phenyl or benzyl, inwhich the phenyl or benzyl group is optionally substituted with halogen,CF), nitro, amino, cyano, hydroxy-C₁₋₄-alkyl, C₁₋₄-alkoxy, sulfonamide,C₁₋₄-alkenyl, C₁₋₄-alkynyl, amino, sulfonyl, sulfonic acid and urea; andR⁴ and R⁵ are each, independently, selected from the group consisting ofhydrogen, halogen, phenoxy, CF₃, nitro, amino, cyano, hydroxyl,C₁₋₄-alkyl, C₁₋₄-alkoxy and phenyl, or a group of the formula —SO₂NR′R″,wherein R′ and R″ independently of each another represents hydrogen orC₁₋₄-alkyl.

In a preferred embodiment of Formula 5, R⁵ is in the 3 position of thearyl ring and R⁴ is in the 2 position of the aryl ring, or R⁴ is in the2 position of the aryl ring and R⁵ is hydrogen.

In one embodiment of Formula 5, R¹ is selected from the group consistingof hydrogen, C₁₋₄-alkyl, C₁₋₄-alkenyl, and C₁₋₄-alkynyl; and R⁴ and R⁵are each, independently, selected from the group consisting of hydrogen,halogen, CF₃, nitro, amino, cyano, hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkoxy,phenoxy and phenyl.

In another embodiment of Formula 5, R¹ is selected from the groupconsisting of hydrogen and C₁₋₄-alkyl; and R⁴ and R⁵ are each,independently, selected from the group consisting of hydrogen, halogen,CF₃, C₁₋₄-alkyl, phenoxy and C₁₋₄-alkoxy.

In another embodiment of Formula 5, R⁴ is C₁₋₄-alkoxy and R⁵ ishydrogen.

In still another embodiment of Formula 5, R¹ is selected from the groupconsisting of —CH₃ and —CH₂CH₃; and R⁴ and R⁵ are each, independently,selected from the group consisting of hydrogen, CH₃, OCH₃ and OEt.

A preferred embodiment of Formula 5 is5-phenyl-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one(Compound H):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 5 is8-ethyl-5-(2-methoxy-phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime (Compound J):

and pharmaceutically acceptable salts thereof.

Another preferred embodiment of Formula 5 is5-(2-ethoxy-phenyl)-8-ethyl-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime (Compound K):

and pharmaceutically acceptable salts thereof.

It is to be understood that all of the compounds of Formulas 1, 2, 3, 4and 5 described above will further include double bonds between adjacentatoms as required to satisfy the valence of each atom. That is, doublebonds are added to provide the following number of total bonds to eachof the following types of atoms: carbon: four bonds; nitrogen: threebonds; oxygen: two bonds; and sulfur: two or six bonds.

It will be noted that the structures of some of the compounds of thisinvention include asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Compounds described herein can be obtained thoughart recognized synthesis strategies.

In one embodiment of the invention, the compounds of the invention thatmodulate the activity of a gated ion channel are capable of chemicallyinteracting with a gated ion channel, including αENaC, βENaC, γENaC,δENaC, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, BLINaC, hINaC,P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, P2X₇, TRPV1, TRPV2, TRPV3, TRPV4,TRPV5, TRPV6. The language “chemical interaction” is intended toinclude, but is not limited to reversible interactions such ashydrophobic/hydrophilic, ionic (e.g., coulombic attraction/repulsion,ion-dipole, charge-transfer), covalent bonding, Van der Waals, andhydrogen bonding. In certain embodiments, the chemical interaction is areversible Michael addition. In a specific embodiment, the Michaeladdition involves, at least in part, the formation of a covalent bond.

In particular embodiment, compound A can be used to treat pain in asubject in need thereof. In one embodiment, the subject is a human.

In another embodiment, compound A can be used to treat inflammation in asubject in need thereof. In one embodiment, the subject is a human.

In another embodiment, compound A is an AMPA antagonist.

Compounds of the inventions can be synthesized according to standardorganic synthesis procedures that are known in the art. Representativesynthesis procedures for compounds similar to the compounds of theinvention can be found in U.S. Pat. No. 5,780,493, U.S. Pat. No.5,843,945, U.S. Pat. No. 6,727,260 and U.S. patent application Ser. Nos.10/737,747 and 11/241,805, each of which are incorporated herein byreference.

Below is a scheme for a specific embodiment of the invention usingorganic starting materials and synthesis procedures well-known inorganic chemistry synthesis:

The synthetic details for the synthesis of Compound A can be found inExample 8.

The end products of the reactions described herein can be isolated byconventional techniques, e.g. by extraction, crystallization,distillation, chromatography, etc.

Acid addition salts of the compounds of the invention are most suitablyformed from pharmaceutically acceptable acids, and include for examplethose formed with inorganic acids e.g. hydrochloric, hydrobromic,sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic,acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g.oxalates can be used for example in the isolation of the compounds ofthe invention, for laboratory use, or for subsequent conversion to apharmaceutically acceptable acid addition salt. Also included within thescope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt isachieved by applying standard techniques, in which an aqueous solutionof the given salt is treated with a solution of base e.g. sodiumcarbonate or potassium hydroxide, to liberate the free base which isthen extracted into an appropriate solvent, such as ether. The free baseis then separated from the aqueous portion, dried, and treated with therequisite acid to give the desired salt.

In vivo hydrolyzable esters or amides of certain compounds of theinvention can be formed by treating those compounds having a freehydroxy or amino functionality with the acid chloride of the desiredester in the presence of a base in an inert solvent such as methylenechloride or chloroform. Suitable bases include triethylamine orpyridine. Conversely, compounds of the invention having a free carboxygroup can be esterified using standard conditions which can includeactivation followed by treatment with the desired alcohol in thepresence of a suitable base.

Examples of pharmaceutically acceptable addition salts include, withoutlimitation, the non-toxic inorganic and organic acid addition salts suchas the hydrochloride derived from hydrochloric acid, the hydrobromidederived from hydrobromic acid, the nitrate derived from nitric acid, theperchlorate derived from perchloric acid, the phosphate derived fromphosphoric acid, the sulphate derived from sulphuric acid, the formatederived from formic acid, the acetate derived from acetic acid, theaconate derived from aconitic acid, the ascorbate derived from ascorbicacid, the benzenesulphonate derived from benzensulphonic acid, thebenzoate derived from benzoic acid, the cinnamate derived from cinnamicacid, the citrate derived from citric acid, the embonate derived fromembonic acid, the enantate derived from enanthic acid, the fumaratederived from fumaric acid, the glutamate derived from glutamic acid, theglycolate derived from glycolic acid, the lactate derived from lacticacid, the maleate derived from maleic acid, the malonate derived frommalonic acid, the mandelate derived from mandelic acid, themethanesulphonate derived from methane sulphonic acid, thenaphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, thephthalate derived from phthalic acid, the salicylate derived fromsalicylic acid, the sorbate derived from sorbic acid, the stearatederived from stearic acid, the succinate derived from succinic acid, thetartrate derived from tartaric acid, the toluene-p-sulphonate derivedfrom p-toluene sulphonic acid, and the like. Particularly to preferredsalts are sodium, lysine and arginine salts of the compounds of theinvention. Such salts can be formed by procedures well known anddescribed in the art.

Other acids such as oxalic acid, which can not be consideredpharmaceutically acceptable, can be useful in the preparation of saltsuseful as intermediates in obtaining a chemical compound of theinvention and its pharmaceutically acceptable acid addition salt.

Metal salts of a chemical compound of the invention includes alkalimetal salts, such as the sodium salt of a chemical compound of theinvention containing a carboxy group.

In the context of this invention the “onium salts” of N-containingcompounds are also contemplated as pharmaceutically acceptable salts.Preferred “onium salts” include the alkyl-onium salts, thecycloalkyl-onium salts, and the cycloalkyl-onium salts.

The chemical compound of the invention can be provided in dissoluble orindissoluble forms together with a pharmaceutically acceptable solventssuch as water, ethanol, and the like. Dissoluble forms can also includehydrated forms such as the monohydrate, the dihydrate, the hemihydrate,the trihydrate, the tetrahydrate, and the like. In general, thedissoluble forms are considered equivalent to indissoluble forms for thepurposes of this invention.

A. Stereoisomers

The chemical compounds of the present invention can exist in (+) and (−)forms as well as in racemic forms. The racemates of these isomers andthe individual isomers themselves are within the scope of the presentinvention.

Racemic forms can be resolved into the optical antipodes by knownmethods and techniques. One way of separating the diastereomeric saltsis by use of an optically active acid, and liberating the opticallyactive amine compound by treatment with a base. Another method forresolving racemates into the optical antipodes is based uponchromatography on an optical active matrix. Racemic compounds of thepresent invention can thus be resolved into their optical antipodes,e.g., by fractional crystallization of d- or l-(tartrates, mandelates,or camphorsulphonate) salts for example.

The chemical compounds of the present invention can also be resolved bythe formation of diastereomeric amides by reaction of the chemicalcompounds of the present invention with an optically active activatedcarboxylic acid such as that derived from (+) or (−) phenylalanine, (+)or (−) phenylglycine, (+) or (−) camphanic acid or by the formation ofdiastereomeric carbamates by reaction of the chemical compound of thepresent invention with an optically active chloroformate or the like.

Additional methods for the resolving the optical isomers are known inthe art. Such methods include those described by Jaques J, Collet A, andWilen S in “Enantiomers, Racemates, and Resolutions”, John Wiley andSons, New York (1981).

Optical active compounds can also be prepared from optical activestarting materials.

Moreover, some of the chemical compounds of the invention being oximes,can thus exist in two forms, syn- and anti-form (Z- and E-form),depending on the arrangement of the substituents around the —C═N— doublebond. A chemical compound of the present invention can thus be the syn-or the anti-form (Z- and E-form), or it can be a mixture hereof. It isto be understood that both the syn- and anti-form (Z- and E-form) of aparticular compound is within the scope of the present invention, evenwhen the compound is represented herein (i.e., through nomenclature orthe actual drawing of the molecule) in one form or the other.

In yet another embodiment, the invention pertains to pharmaceuticalcompositions comprising gated ion channel modulating compounds describedherein and a pharmaceutical acceptable carrier.

In another embodiment, the invention includes any novel compound orpharmaceutical compositions containing compounds of the inventiondescribed herein. For example, compounds and pharmaceutical compositionscontaining compounds set forth herein (e.g., compounds of the invention)are part of this invention, including salts thereof, e.g.,pharmaceutically acceptable salts.

Assays

The present invention relates to a method of modulating gated ionchannel activity. As used herein, the various forms of the term“modulate” include stimulation (e.g., increasing or upregulating aparticular response or activity) and inhibition (e.g., decreasing ordownregulating a particular response or activity). In one aspect, themethods of the present invention comprise contacting a cell with aneffective amount of a gated ion channel modulator compound, e.g. acompound of the invention, thereby modulating the activity of a gatedion channel. In certain embodiments, the effective amount of thecompound of the invention inhibits the activity of the gated ion channel

The gated ion channels of the present invention are comprised of atleast one subunit belonging to the DEG/ENaC, TRPV (also referred to asvanilloid) and/or P2X gene superfamilies. In one aspect the gated ionchannel is comprised of at least one subunit selected from the groupconsisting of αENaC, βENaC, γENaC, δENaC, ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3, ASIC4, BLINaC, hINaC, P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆,P2X₇, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6. In one aspect, theDEG/ENaC gated ion channel is comprised of at least one subunit selectedfrom the group consisting of αENaC, βENaC, γENaC, δENaC, BLINaC, hINaC,ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4. In certainembodiments, the DEG/ENaC gated ion channel is comprised of at least onesubunit selected from the group consisting of ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3, and ASIC4. In certain embodiments, the gated ion channelis comprised of ASIC1a, ASIC1b, or ASIC3. In another aspect of theinvention, P2X gated ion channel is comprised of at least one subunitselected from the group consisting of P2X₁, P2X₂, P2X₃, P2X₄, P2X₅,P2X₆, and P2X₇. In yet another aspect of the invention, the TRPV gatedion channel is comprised of at least one subunit selected from the groupTRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6. In another aspect, thegated ion channel is a heteromultimeric gated ion channel, including,but not limited to, αENaC, βENaC and γENaC; αENaC, βENaC and δENaC;ASIC1a and ASIC2a; ASIC 1a and ASIC2b; ASIC1a and ASIC3; ASIC1b andASIC3; ASIC2a and ASIC2b; ASIC2a and ASIC3; ASIC2b and ASIC3; ASIC1a,ASIC2a and ASIC3; ASIC3 and P2X, e.g. P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆and P2X₇, preferably ASIC3 and P2X₂; ASIC3 and P2X₃; and ASIC3, P2X₂ andP2X₃; ASIC4 and at least one of ASIC1a, ASIC1b, ASIC2a, ASIC2b, andASIC3; BLINaC (or hINaC) and at least one of ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3, and ASIC4; 3ENaC and ASIC, e.g. ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3 and ASIC4; P2X₁ and P2X₂, P2X₁ and P2X₅, P2X₂ and P2X₃,P2X₂ and P2X₆, P2X₄ and P2X₆, TRPV1 and TRPV2, TRPV5 and TRPV6, TRPV1and TRPV4.

Assays for determining the ability of a compound within the scope of theinvention to modulate the activity of gated ion channels are well knownin the art and described herein in the Examples section. Other assaysfor determining the ability of a compound to modulate the activity of agated ion channel are also readily available to the skilled artisan.

The gated ion channel modulating compounds of the invention can beidentified using the following screening method, which method comprisesthe subsequent steps of

(i) subjecting a gated ion channel containing cell to the action of aselective activator, e.g., protons by adjustment of the pH to an acidiclevel, ATP by diluting sufficient amounts of ATP in the perfusion bufferor temperature by heating the perfusion buffer to temperatures above 37°C.;

(ii) subjecting a gated ion channel containing cell to the action of thechemical compound (the compound can be co-applied, pre-applied orpost-applied); and

(iii) monitoring the change in membrane potential or ionic currentinduced by the activator, e.g., protons, on the gated ion channelcontaining cell. Alternatively, fluorescent imaging can be utilized tomonitor the effect induced by the activator, e.g., protons, on the gatedion channel containing cell.

The gated ion channel containing cells can be subjected to the action ofprotons by adjustment of the pH to an acidic level using any convenientacid or buffer, including organic acids such as formic acid, aceticacid, citric acid, ascorbic acid, 2-morpholinoethanesulfonic acid (MES)and lactic acid, and inorganic acids such as hydrochloric acid,hydrobromic acid and nitric acid, perchloric acid and phosphoric acid.

In the methods of the invention, the current flux induced by theactivator, e.g., protons, across the membrane of the gated ion channelcontaining cell can be monitored by electrophysiological methods, forexample patch clamp or two-electrode voltage clamp techniques.

Alternatively, the change in membrane potential induced by gated ionchannel activators, e.g., protons of the gated ion channel containingcells can be monitored using fluorescence methods. When usingfluorescence methods, the gated ion channel containing cells areincubated with a membrane potential indicating agent that allows for adetermination of changes in the membrane potential of the cells, causedby the added activators, e.g., protons. Such membrane potentialindicating agents include fluorescent indicators, preferably DiBAC₄(3),DiOC5(3), DiOC2(3), DiSBAC2(3) and the FMP (FLIPR membrane potential)dyes (Molecular Devices).

In another alternative embodiment, the change in gated ion channelactivity induced by activators, e.g., protons, on the gated ion channelcan be measured by assessing changes in the intracellular concentrationof certain ions, e.g., calcium, sodium, potassium, magnesium, protons,and chloride in cells by fluorescence. Fluorescence assays can beperformed in multi-well plates using plate readers, e.g., FLIPR assay(Fluorescence Image Plate Reader; available from Molecular Devices),e.g. using fluorescent calcium indicators, e.g. as described in, forexample, Sullivan E., et al. (1999) Methods Mol. Biol. 114:125-33,Jerman, J. C., et al. (2000) Br J Pharmacol 130(4):916-22, and U.S. Pat.No. 6,608,671, the contents of each of which are incorporated herein byreference. When using such fluorescence methods, the gated ion channelcontaining cells are incubated with a selective ion indicating agentthat allows for a determination of changes in the intracellularconcentration of the ion, caused by the added activators, e.g., protons.Such ion indicating agents include fluorescent calcium indicators,preferably Fura-2, Fluo-3, Fluo-4, Fluo4FF, Fluo-5F, Fluo-5N, CalciumGreen, Fura-Red, Indo-1, Indo-5F, and rhod-2, fluorescent sodiumindicators, preferably SBFI, Sodium Green, CoroNa Green, fluorescentpotassium indicators, preferably PBFI, CD222, fluorescent magnesiumindicators, preferably Mag-Fluo-4, Mag-Fura-2, Mag-Fura-5, Mag-Fura-Red,Mag-indo-1, Mag-rho-2, Magnesium Green, fluorescent chloride indicators,preferably SPQ, Bis-DMXPQ, LZQ, MEQ, and MQAE, fluorescent pHindicators, preferably BCECF and BCPCF.

The gated ion channel antagonizing compounds of the invention showactivity in concentrations below 2M, 1.5M, 1M, 500 mM, 250 mM, 100 mM,750 μM, 500 μM, 250 μM, 100 μM, 75 μM, 50 μM, 25 μM, 10 μM, 5 μM, 2.5μM, or below 1 μM. In its most preferred embodiment the ASICantagonizing compounds show activity in low micromolar and the nanomolarrange.

As used herein, the term “contacting” (i.e., contacting a cell e.g. aneuronal cell, with a compound) is intended to include incubating thecompound and the cell together in vitro (e.g., adding the compound tocells in culture) or administering the compound to a subject such thatthe compound and cells of the subject are contacted in vivo. The term“contacting” is not intended to include exposure of cells to a modulatoror compound that can occur naturally in a subject (i.e., exposure thatcan occur as a result of a natural physiological process).

A. In Vitro Assays

Gated ion channel polypeptides for use in the assays described hereincan be readily produced by standard biological techniques or by chemicalsynthesis. For example, a host cell transfected with an expressionvector containing a nucleotide sequence encoding the desired gated ionchannel can be cultured under appropriate conditions to allow expressionof the peptide to occur. Alternatively, the gated ion channel can beobtained by culturing a primary cell line or an established cell linethat can produce the gated ion channel.

The methods of the invention can be practiced in vitro, for example, ina cell-based culture screening assay to screen compounds whichpotentially bind, activate or modulate gated ion channel function. Insuch a method, the modulating compound can function by interacting withand eliminating any specific function of gated ion channel in the sampleor culture. The modulating compounds can also be used to control gatedion channel activity in neuronal cell culture.

Cells for use in in vitro assays, in which gated ion channels arenaturally present, include various cells, such as cortical neuronalcells, in particular mouse or rat cortical neuronal cells, and humanembryonic kidney (HEK) cells, in particular the HEK293 cell line. Forexample, cells can be cultured from embryonic human cells, neonatalhuman cells, and adult human cells. Primary cell cultures can also beused in the methods of the invention. For example, sensory neuronalcells can also be isolated and cultured in vitro from different animalspecies. The most widely used protocols use sensory neurons isolatedfrom neonatal (Eckert, et al. (1997) J Neurosci Methods 77:183-190) andembryonic (Vasko, et al. (1994) J Neurosci 14:4987-4997) rat. Trigeminaland dorsal root ganglion sensory neurons in culture exhibit certaincharacteristics of sensory neurons in vivo.

Alternatively, the gated ion channel, e.g., a gated channel, e.g., aproton gated ion channel, can be exogenous to the cell in question, andcan in particular be introduced by recombinant DNA technology, such astransfection, microinjection or infection. Such cells include Chinesehamster ovary (CHO) cells, HEK cells, African green monkey kidney cellline (CV-1 or CV-1-derived COS cells, e.g. COS-1 and COS-7) Xenopuslaevis oocytes, or any other cell lines capable of expressing gated ionchannels.

The nucleotide and amino acid sequences of the gated ion channels of theinvention are known in the art. For example, the sequences of the humangated channels can be found in Genbank GI Accession Nos: GI:40556387(ENaCalpha Homo sapiens); GI:4506815 (ENaCalpha Homo sapiens);GI:4506816 (ENaCbeta Homo sapiens); GI:4506817 (ENaCbeta Homo sapiens);GI:34101281 (ENaCdelta Homo sapiens); GI:34101282 (ENaCdelta Homosapiens); GI:42476332 (ENaCgamma Homo sapiens); GI:42476333 (ENaCgammaHomo sapiens); GI:31442760 (HINAC Homo sapiens); GI:31442761 (HINAC Homosapiens); GI: 21536350 (ASIC1a Homo sapiens); GI:21536351 (ASIC1a Homosapiens); GI:21536348(ASIC1b Homo sapiens); GI:21536349 (ASIC1b Homosapiens); GI:34452694 (ASIC2; transcript variant 1 Homo sapiens);GI:34452695 (ASIC2; isoform 1 Homo sapiens); GI:34452696(ASIC2;transcript variant 2 Homo sapiens); GI:9998944 (ASIC2; isoform 2 Homosapiens); GI:4757709 (ASIC3; transcript variant 1 Homo sapiens);GI:4757710(ASIC3; isoform 1 Homo sapiens); GI:9998945(ASIC3; transcriptvariant 2 Homo sapiens); GI:9998946 (ASIC3; isoform 2 Homo sapiens);GI:9998947 (ASIC3; transcript variant 3 Homo sapiens); GI: 9998948(ASIC3; isoform 3 Homo sapiens); GI:33519441 (ASIC4; transcript variant1 Homo sapiens); GI:33519442 (ASIC4; isoform 1 Homo sapiens);GI:33519443 (ASIC4; transcript variant 2 Homo sapiens); GI:33519444(ASIC4; isoform 2 Homo sapiens); GI:27894283 (P2X₁ Homo sapiens);GI:4505545 (P2X₁ Homo sapiens); GI:28416917 (P2X₂; transcript variant 1Homo sapiens); GI:25092719 (P2X₂; isoform A Homo sapiens); GI:28416922(P2X₂; transcript variant 2 Homo sapiens);

GI:28416923 (P2X₂; isoform B Homo sapiens); GI:28416916(P2X₂; transcriptvariant 3 Homo sapiens); GI:7706629 (P2X₂; isoform C Homo sapiens);GI:28416918(P2X₂; transcript variant 4 Homo sapiens); GI:25092733 (P2X₂;isoform D Homo sapiens); GI:28416920 (P2X₂; transcript variant 5 Homosapiens); GI:28416921 (P2X₂; isoform H Homo sapiens); GI:28416919 (P2X₂;transcript variant 6 Homo sapiens); GI:27881423 (P2X₂; isoform I Homosapiens); GI:28416924 (P2X₃ Homo sapiens); GI:28416925 (P2X₃ Homosapiens); GI:28416926 (P2X₄; transcript variant 1 Homo sapiens);GI:28416927 (P2X₄; isoform A Homo sapiens); GI: 28416928 (P2X₄;transcript variant 2 Homo sapiens); GI:28416929 (P2X₄; isoform B Homosapiens); GI:28416930 (P2X₄; transcript variant 3 Homo sapiens);GI:28416931 (P2X₄; isoform C Homo sapiens); GI:28416932 (P2X₅;transcript variant 1 Homo sapiens); GI:28416933 (P2X₅; isoform A Homosapiens); GI:28416934 (P2X₅; transcript variant 2 Homo sapiens);GI:28416935 (P2X₅; isoform B Homo sapiens); GI:28416936 (P2X₅;transcript variant 3 Homo sapiens); GI:28416937 (P2X₅; isoform C Homosapiens); GI:38327545 (P2X₆ Homo sapiens); GI:4885535 (P2X₆ Homosapiens); GI:34335273 (P2X₇; transcript variant 1 Homo sapiens);GI:29294631 (P2X₇; isoform A Homo sapiens); GI:34335274 (P2X₇;transcript variant 2 Homo sapiens); in GI:29294633 (P2X₇; isoform B Homosapiens); GI:18375666 (TRPV1; transcript variant 1 Homo sapiens);GI:18375667(TRPV1; vanilloid receptor subtype 1 Homo sapiens);GI:18375664 (TRPV1; transcript variant 2 Homo sapiens); GI:18375665(TRPV1; vanilloid receptor subtype 1 Homo sapiens); GI:18375670 (TRPV1;transcript variant 3 Homo sapiens); GI:18375671(TRPV1; vanilloidreceptor subtype 1 Homo sapiens); GI:18375668 (TRPV1; transcript variant4 Homo sapiens); GI:18375669 (TRPV1; vanilloid receptor subtype 1 Homosapiens); GI:7706764 (VRL-1; transcript variant 1 Homo sapiens);GI:7706765 (VRL-1; vanilloid receptor-like protein 1 Homo sapiens);GI:22547178 (TRPV2; transcript variant 2 Homo sapiens); GI:20127551(TRPV2; vanilloid receptor-like protein 1 Homo sapiens); GI:22547183(TRPV4; transcript variant 1 Homo sapiens); GI:22547184 (TRPV4; isoformA Homo sapiens); GI:22547179 (TRPV4; transcript variant 2 Homo sapiens);GI:22547180 (TRPV4; isoform B Homo sapiens); GI:21361832 (TRPV5 Homosapiens); GI:17505200 (TRPV5 Homo sapiens); GI:21314681 (TRPV6 Homosapiens); GI:21314682 (TRPV6 Homo sapiens); GI: 34452696 (ACCN1;transcript variant 2; Homo sapiens). The contents of each of theserecords are incorporated herein by reference. Additionally, sequencesfor channels of other species are readily available and obtainable bythose skilled in the art.

A nucleic acid molecule encoding a gated ion channel for use in themethods of the present invention can be amplified using cDNA, mRNA, orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis. Using all or a portion of such nucleic acidsequences, nucleic acid molecules of the invention can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Expression vectors, containing a nucleic acid encoding a gated ionchannel, e.g., a gated ion channel subunit protein, e.g., αENaC, βENaC,γENaC, δENaC, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, BLINaC,hINaC P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, P2X₇, TRPV1, TRPV2, TRPV3,TRPV4, TRPV5, and TRPV6 protein (or a portion thereof) are introducedinto cells using standard techniques and operably linked to regulatorysequence. Such regulatory sequences are described, for example, inGoeddel, Methods in Enzymology: Gene Expression Technology vol. 185,Academic Press, San Diego, Calif. (1991). Regulatory sequences includethose which direct constitutive expression of a nucleotide sequence inmany types of host cell and those which direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences). It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the host cell to be transformed, the level ofexpression of protein desired, and the like. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

Examples of vectors for expression in yeast S. cerevisiae includepYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

Examples of mammalian expression vectors include pCDM8 (Seed, 1987,Nature 329:840), pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195),pcDNA3. When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. For other suitable expressionsystems for eukaryotic cells see chapters 16 and 17 of Sambrook et al.

B. In Vivo Assays

The activity of the compounds of the invention as described herein tomodulate one or more gated ion channel activities (e.g., a gated ionchannel modulator, e.g., a compound of the invention) can be assayed inan animal model to determine the efficacy, toxicity, or side effects oftreatment with such an agent. Alternatively, an agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent.

Animal models for determining the ability of a compound of the inventionto modulate a gated ion channel biological activity are well known andreadily available to the skilled artisan. Examples of animal models forpain and inflammation include, but are not limited to the models listedin Table 1. Animal models for investigating neurological disordersinclude, but are not limited to, those described in Morris et al.,(Learn. Motiv. 1981; 12: 239-60) and Abeliovitch et al., Cell 1993; 75:1263-71). An example of an animal model for investigating mental andbehavioral disorders is the Geller-Seifter paradigm, as described inPsychopharmacology (Berl). 1979 Apr. 11; 62(2):117-21.

Genitourinary models include methods for reducing the bladder capacityof test animals by infusing either protamine sulfate and potassiumchloride (See, Chuang, Y. C. et al., Urology 61(3): 664-670 (2003)) ordilute acetic acid (See, Sasaki, K. et al., J. Urol. 168(3): 1259-1264(2002)) into the bladder. For urinary tract disorders involving thebladder using intravesically administered protamine sulfate as describedin Chuang et al. (2003) Urology 61: 664-70. These methods also includethe use of a well accepted model of for urinary tract disordersinvolving the bladder using intravesically administered acetic acid asdescribed in Sasaki et al. (2002) J. Urol. 168: 1259-64. Efficacy fortreating spinal cord injured patients can be tested using methods asdescribed in Yoshiyama et al. (1999) Urology 54: 929-33.

Animal models of neuropathic pain based on injury inflicted to a nerve(mostly the sciatic nerve) are described in Bennett et al., 1988, Pain33:87-107; Seltzer et al., 1990, Pain 43:205-218; Kim et al., 1992, Pain50:355-363; Decosterd et al., 2000, Pain 87:149-158 and DeLeo et al.,1994, Pain 56:9-16. There are also models of diabetic neuropathy (STZinduced diabetic neuropathy—Courteix et al., 1994, Pain 57:153-160) anddrug induced neuropathies (vincristine induced neuropathy—Aley et al.,1996, Neuroscience 73: 259-265; oncology-related immunotherapy, anti-GD2antibodies—Slart et al., 1997, Pain 60:119-125). Acute pain in humanscan be reproduced using in murine animals chemical stimulation: Martinezet al., Pain 81: 179-186; 1999 (the writhing test—intraperitoneal aceticacid in mice), Dubuisson et al. Pain 1977; 4: 161-74 (intraplantarinjection of formalin). Other types of acute pain models are describedin Whiteside et al., 2004, Br J Pharmacol 141:85-91 (the incisionalmodel, a post-surgery model of pain) and Johanek and Simone, 2004, Pain109:432-442 (a heat injury model). An animal model of inflammatory painusing complete Freund's adjuvant (intraplantar injection) is describedin Jasmin et al., 1998, Pain 75: 367-382. Intracapsular injection ofirritant agents (complete Freund's adjuvant, iodoacetate, capsaicine,urate crystals, etc.) is used to develop arthritis models in animals(Fernihough et al., 2004, Pain 112:83-93; Coderre and Wall, 1987, Pain28:379-393; Otsuki et al., 1986, Brain Res. 365:235-240). Astress-induced hyperalgesia model is described in Quintero et al., 2000,Pharmacology, Biochemistry and Behavior 67:449-458. Further animalmodels for pain are considered in an article of Walker et al. 1999Molecular Medicine Today 5:319-321, comparing models for different typesof pain, which are acute pain, chronic/inflammatory pain andchronic/neuropathic pain, on the basis of behavioral signs. Animalmodels for depression are described by E. Tatarczynska et al., Br. J.Phainiacol. 132(7): 1423-1430 (2001) and P. J. M. Will et al., Trends inPharmacological Sciences 22(7):331-37 (2001)); models for anxiety aredescribed by D. Treit, “Animal Models for the Study of Anti-anxietyAgents: A Review,” Neuroscience & Biobehavioral Reviews 9(2):203-222(1985). Additional animal models for pain are also described herein inthe Exemplification section.

Gastrointestinal models can be found in: Gawad, K. A., et al.,Ambulatory long-term pH monitoring in pigs, Surg Endosc, (2003);Johnson, S. E. et al., Esophageal Acid Clearance Test in Healthy Dogs,Can. J. Vet. Res. 53(2): 244-7 (1989); and Cicente, Y. et al.,Esophageal Acid Clearance: More Volume-dependent Than Motility Dependentin Healthy Piglets, J. Pediatr. Gastroenterol. Nutr. 35(2): 173-9(2002). Models for a variety of assays can be used to assessvisceromotor and pain responses to rectal distension. See, for example,Gunter et al., Physiol. Behay., 69(3): 379-82 (2000), Depoortere et al.,J. Pharmacol. and Exp. Ther., 294(3): 983-990 (2000), Morteau et al.,Fund. Clin. Pharmacol., 8(6): 553-62 (1994), Gibson et al.,Gastroenterology (Suppl. 1), 120(5): A19-A20 (2001) and Gschossmann etal., Eur. J. Gastro. Hepat., 14(10): 1067-72 (2002) the entire contentsof which are each incorporated herein by reference.

Gastrointestinal motility can be assessed based on either the in vivorecording of mechanical or electrical events associated intestinalmuscle contractions in whole animals or the activity of isolatedgastrointestinal intestinal muscle preparations recorded in vitro inorgan baths (see, for example, Yaun et al., Br. J. Pharmacol.,112(4).1095-1100 (1994), Jin et al., J. Pharm. Exp. Ther., 288(1): 93-97(1999) and Venkova et al., J. Pharm. Exp. Ther., 300(3): 1046-1052(2002)). Tatersall et al. and Bountra et al., European Journal ofPharmacology, 250: (1993) R5 and 249:(1993) R3-R4 and Milano et al., J.Pharmacol. Exp. Ther., 274(2): 951-961 (1995).

TABLE 1 Non-limiting examples of potential Modality clinical indicationsModel Name tested Brief Description (Reference) ACUTE PHASIC PAINTail-flick Thermal Tip of tail of rats is immersed if hot water and timeAcute nociceptive pain to withdrawal from water is measured.Alternatively, (Hardy et al. Am J Physiol 1957; 189: a radiant heatsource is applied to the tail and time 1-5.; Ben-Bassat et al. ArchIntern to withdrawal is determined. Analgesic effect is Pharmacodyn Ther1959; 122: 434-47.) evidenced by a prolongation of the latency periodhot-plate Thermal Rats walk over a heated surface with increasing Acutenociceptive pain temperature and observed for specific nociceptive(Woolfe et al. J Pharmacol Exp Ther behavior such paw licking, jumping.Time to 1944; 80: 300-7.) appearance of such behavior is measured.Analgesic effects are evidenced by a prolonged latency. HargreavesThermal A focused beam of light is projected onto a small Acutenociceptive pain Test surface of the hind leg of a rat with increasing(Yeomans et al. Pain 1994; 59: 85-94.) temperature. Time to withdrawalis measured. Analgesic effect translates into a prolonged latency PinTest or Mechanical An increasing calibrated pressure is applied to theAcute nociceptive pain Randall Selitto paw of rats with a blunt pin.Pressure intensity is (Green et al. Br J Pharmacol 1951; 6: measured.Alternatively increased pressure is 572-85.; Randall et al. Arch Intapplied to the paw using a caliper until pain Pharmacodyn Ther 1957;111: 409-19) threshold is reached and animals withdraw the paw.HYPERALGESIA MODELS/CHRONIC INFLAMMATORY PAIN MODELS Hargreaves orThermal A sensitizing agent (e.g, complete Freund's Chronic painassociated with tissue Randal & and/or adjuvant (CFA), carrageenin,turpentine etc.) is inflammation, e.g. post-surgical pain, Selittomechanical injected into the paw of rats creating a local (Hargreaves etal. Pain 1988; 32: 77-88.) inflammation and sensitivities to mechanicalRandall LO, Selitto JJ. Arch Int (Randall & Selitto) and/or therma(Hargreaves)I Pharmacodyn1957; 3: 409-19. stimulation are measured withcomparison to the contralateral non-sensitized paw Yeomans Thermal Rathind paw in injected with capsaicin, a Chronic pain associated withtissue model sensitizing agent for small C-fibers or DMSO, ainflammation, e.g. post-surgical pain sensitizing agent for A-deltafibers. A radiant heat is (Yeomans et al. Pain 1994; 59: 85-94.; appliedwith different gradient to differentially Otsuki et al. Brain Res 1986;365: stimulate C-fibers or A-delta fibers and discriminate 235-240.)between the effects mediated by both pathways CHRONIC MALIGNANT PAIN(CANCER PAIN) Bone Cancer Thermal In this model, osteolytic mousesarcoma Bone cancer pain Model and/or NCTC2472 cells are used to inducebone cancer by (Schwei et al., J. Neurosci. 1999; 19: mechanicalinjecting tumor cells into the marrow space of the 10886-10897.) femurbone and sealing the injection site Cancer Thermal Meth A sarcoma cellsare implanted around the Malignant neuropathic pain invasion pain and/orsciatic nerve in BALB/c mice and these animals (Shimoyama et al., Pain2002; 99: model (CIP) mechanical develop signs of allodynia and thermalhyperalgesia 167-174.) as the tumor grows, compressing the nerve.Spontaneous pain (paw lifting) is also visible. CHRONIC NON-MALIGNANTPAIN Muscle Pain Thermal Repeated injections of acidic saline into oneFibromyalgia and/or gastrocnemius muscle produces bilateral, long-(Sluka et al. Pain 2003; 106: 229-239.) mechanical lasting mechanicalhypersensitivity of the paw (i.e. hyperalgesia) without associatedtissue damage UV-irradiation Thermal Exposure of the rat hind paw to UVirradiation Inflammatory pain associated with first- and/or produceshighly reliable and persistent allodynia. and second-degree burns.mechanical Various irradiation periods with UV-B produce skin (Perkinset al. Pain 1993; 53: 191-197.) inflammation with different time coursesCHRONIC NEUROPATHIC PAIN Chronic Mostly Loose chronic ligature of thesciatic nerve. Thermal Clinical Neuropathic pain: nerve Constrictionmechanical or mechanical sensitivities are tested using Von compressionand direct mechanical Injury (CCI) or but aso Frey hairs or the pawwithdrawal test (Hargreaves) neuronal damage might be relevant Bennettand thermal clinical comparisons Xie model (Bennett & Xie,Neuropharmacology 1984; 23: 1415-1418.) Chung's Mostly Tight ligation ofone of the two spinal nerves of the Same as above: root compressionmodel or mechanical sciatic nerve. Thermal or mechanical sensitivitiesmight be a relevant clinical comparison Spinal Nerve but aso are testedusing Von Frey hairs or the paw (Kim and Chung, Pain 1990; 41: 235-251.)Ligation thermal withdrawal test (Hargreaves) model (SNL)

Alternatively, the compounds can also be assayed in non-human transgenicanimals containing exogenous sequences encoding one or more gated ionchannels. As used herein, a “transgenic animal” is a non-human animal,preferably a mammal, more preferably a rodent such as a rat or mouse, inwhich one or more of the cells of the animal includes a transgene. Otherexamples of transgenic animals include non-human primates, sheep, dogs,cows, goats, chickens, amphibians, etc. Methods for generatingtransgenic animals via embryo manipulation and microinjection,particularly animals such as mice, have become conventional in the artand are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986. Similar methods are used for production of other transgenicanimals.

A homologous recombinant animal can also be used to assay the compoundsof the invention. Such animals can be generated according to well knowntechniques (see, e.g., Thomas and Capecchi, 1987, Cell 51:503; Li etal., 1992, Cell 69:915; Bradley, Teratocarcinomas and Embryonic StemCells: A Practical Approach, Robertson, Ed., IRL, Oxford, 1987, pp.113-152;Bradley (1991) Current Opinion in Bio/Technology 2:823-829 andin PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO93/04169).

Other useful transgenic non-human animals can be produced which containselected systems which allow for regulated expression of the transgene(see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA89:6232-6236). Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O′Gorman et al., 1991,Science 251:1351-1355).

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically (or prophylactically) effectiveamount of a gated ion channel modulator, and preferably one or morecompounds of the invention described above, and a pharmaceuticallyacceptable carrier or excipient. Suitable pharmaceutically acceptablecarriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the subject. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose, dextrose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose,methylcellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, castor oil, tetraglycol, and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate, esters ofpolyethylene glycol and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide, sodium hydroxide, potassium hydroxide, carbonates,triethylanolamine, acetates, lactates, potassium citrate and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol and derivatives such as vitamin E tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, sodium citrate and the like.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, cyclodextrin,magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil,fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc.The pharmaceutical preparations can be sterilized and if desired, mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, flavoring and/or aromatic substances and the likewhich do not deleteriously react with the active compounds. Thepharmaceutically acceptable carriers can also include atonicity-adjusting agent such as dextrose, glycerine, mannitol andsodium chloride.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,polyvinyl pyrollidone, sodium saccharine, cellulose, magnesiumcarbonate, etc.

The composition can be formulated in accordance with the routineprocedures as a pharmaceutical composition adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition can also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampule orsachet indicating the quantity of active agent. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water, saline ordextrose/water. Where the composition is administered by injection, anampule of sterile water for injection or saline can be provided so thatthe ingredients can be mixed prior to administration.

The pharmaceutical compositions of the invention can also include anagent which controls release of the gated ion channel modulatorcompound, thereby providing a timed or sustained release composition.

The present invention also relates to prodrugs of the gated ion channelmodulators disclosed herein, as well as pharmaceutical compositionscomprising such prodrugs. For example, compounds of the invention whichinclude acid functional groups or hydroxyl groups can also be preparedand administered as a corresponding ester with a suitable alcohol oracid. The ester can then be cleaved by endogenous enzymes within thesubject to produce the active agent.

Formulations of the present invention include those suitable for oral,nasal, topical, mucous membrane, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations canconveniently be presented in unit dosage form and can be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound thatproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration can be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention can also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions can also comprise bufferingagents. Solid compositions of a similar type can also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, can optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They can also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They can be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions can also optionally containopacifying agents and can be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms can contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration can be presented as a suppository,which can be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound canbe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that can berequired.

The ointments, pastes, creams and gels can contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which can be reconstituted into sterileinjectable solutions or dispersions just prior to use, which can containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that can beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It can also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form can be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

Methods of Administration

The invention provides a method of treating a condition mediated bygated ion channel activity in a subject, including, but not limited to,pain, inflammatory disorders, neurological disorders, gastrointestinaldisorders and genitourinary disorders. The method comprises the step ofadministering to the subject a therapeutically effective amount of agated ion channel modulator. The condition to be treated can be anycondition which is mediated, at least in part, by the activity of agated ion channel (e.g., ASIC1a and/or ASIC3).

The quantity of a given compound to be administered will be determinedon an individual basis and will be determined, at least in part, byconsideration of the individual's size, the severity of symptoms to betreated and the result sought. The gated ion channel activity modulatorsdescribed herein can be administered alone or in a pharmaceuticalcomposition comprising the modulator, an acceptable carrier or diluentand, optionally, one or more additional drugs.

These compounds can be administered to humans and other animals fortherapy by any suitable route of administration. The gated ion channelmodulator can be administered subcutaneously, intravenously,parenterally, intraperitoneally, intradermally, intramuscularly,topically, enterally (e.g., orally), rectally, nasally, buccally,sublingually, systemically, vaginally, by inhalation spray, by drug pumpor via an implanted reservoir in dosage formulations containingconventional non-toxic, physiologically acceptable carriers or vehicles.The preferred method of administration is by oral delivery. The form inwhich it is administered (e.g., syrup, elixir, capsule, tablet,solution, foams, emulsion, gel, sol) will depend in part on the route bywhich it is administered. For example, for mucosal (e.g., oral mucosa,rectal mucosa, intestinal mucosa, bronchial mucosa) administration, nosedrops, aerosols, inhalants, nebulizers, eye drops or suppositories canbe used. The compounds and agents of this invention can be administeredtogether with other biologically active agents, such as analgesics,e.g., opiates, anti-inflammatory agents, e.g., NSAIDs, anesthetics andother agents which can control one or more symptoms or causes of a gatedion channel mediated condition.

In a specific embodiment, it can be desirable to administer the agentsof the invention locally to a localized area in need of treatment; thiscan be achieved by, for example, and not by way of limitation, localinfusion during surgery, topical application, transdermal patches, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes orfibers. For example, the agent can be injected into the joints or theurinary bladder.

The compounds of the invention can, optionally, be administered incombination with one or more additional drugs which, for example, areknown for treating and/or alleviating symptoms of the condition mediatedby a gated ion channel (e.g., ASIC1a and/or ASIC3). The additional drugcan be administered simultaneously with the compound of the invention,or sequentially. For example, the compounds of the invention can beadministered in combination with at least one of an analgesic, ananti-inflammatory agent, an anesthetic, a corticosteroid (e.g.,dexamethasone, beclomethasone diproprionate (BDP) treatment), ananti-convulsant, an antidepressant, an anti-nausea agent, ananti-psychotic agent, a cardiovascular agent (e.g., a beta-blocker) or acancer therapeutic. In certain embodiments, the compounds of theinvention are administered in combination with a pain drug. As usedherein the phrase, “pain drugs” is intended to refer to analgesics,anti-inflammatory agents, anesthetics, corticosteroids, antiepileptics,barbiturates, antidepressants, and marijuana.

The combination treatments mentioned above can be started prior to,concurrent with, or after the administration of the compositions of thepresent invention. Accordingly, the methods of the invention can furtherinclude the step of administering a second treatment, such as a secondtreatment for the disease or disorder or to ameliorate side effects ofother treatments. Such second treatment can include, e.g.,anti-inflammatory medication and any treatment directed toward treatingpain. Additionally or alternatively, further treatment can includeadministration of drugs to further treat the disease or to treat a sideeffect of the disease or other treatments (e.g., anti-nausea drugs,anti-inflammatory drugs, anti-depressants, anti-psychiatric drugs,anti-convulsants, steroids, cardiovascular drugs, and cancerchemotherapeutics).

As used herein, an “analgesic” is an agent that relieves or reduces painor any signs or symptoms thereof (e.g., hyperalgesia, allodynia,dysesthesia, hyperesthesia, hyperpathia, paresthesia) and can alsoresult in the reduction of inflammation, e.g., an anti-inflammatoryagent. Analgesics can be subdivided into NSAIDs(non-steroidal-anti-inflammatory drugs), narcotic analgesics, includingopioid analgesics, and non-narcotic analgesics. NSAIDs can be furthersubdivided into non-selective COX (cyclooxygenase) inhibitors, andselective COX2 inhibitors. Opioid analgesics can be natural, syntheticor semi-synthetic opioid analgesics, and include for example, morphine,codeine, meperidine, propxyphen, oxycodone, hydromorphone, heroine,tramadol, and fentanyl. Non-narcotic analgesics (also called non-opioid)analgesics include, for example, acetaminophen, clonidine, NMDAantagonists, vanilloid receptor antagonists (e.g., TRPV1 antagonists),pregabalin, endocannabinoids and cannabinoids. Non-selective COXinhibitors include, but are not limited to acetylsalicylic acid (ASA),ibuprofen, naproxen, ketoprofen, piroxicam, etodolac, and bromfenac.Selective COX2 inhibitors include, but are not limited to celecoxib,valdecoxib, parecoxib, and etoricoxib.

As used herein an “anesthetic” is an agent that interferes with senseperception near the site of administration, a local anesthetic, orresult in alteration or loss of consciousness, e.g., systemic anestheticagents. Local anesthetics include but are not limited to lidocaine andbuvicaine.

Non-limiting examples of antiepileptic agents are carbamazepine,phenyloin and gabapentin. Non-limiting examples of antidepressants areamitriptyline and desmethylimiprimine.

Non-limiting examples of anti-inflammatory drugs include corticosteroids(e.g., hydrocortisone, cortisone, prednisone, prednisolone, methylprednisone, triamcinolone, fluprednisolone, betamethasone anddexamethasone), salicylates, NSAIDs, antihistamines and H₂ receptorantagonists.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe subject's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

Regardless of the route of administration selected, the compounds of thepresent invention, which can be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention can be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the subject being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, dosages of a compound of theinvention can be determined by deriving dose-response curves using ananimal model for the condition to be treated. For example, the physicianor veterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous and subcutaneousdoses of the compounds of this invention for a subject, when used forthe indicated analgesic effects, will range from about 0.0001 to about100 mg per kilogram of body weight per day, more preferably from about0.01 to about 100 mg per kg per day, and still more preferably fromabout 1.0 to about 50 mg per kg per day. An effective amount is thatamount treats a gated ion channel-associated state or gated ion channeldisorder.

If desired, the effective daily dose of the active compound can beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

Methods of Treatment

The above compounds can be used for administration to a subject for themodulation of a gated ion channel-mediated activity, involved in, butnot limited to, pain, inflammatory disorders, neurological disorders,and any abnormal function of cells, organs, or physiological systemsthat are modulated, at least in part, by a gated ion channel-mediatedactivity. Additionally, it is understood that the compounds can alsoalleviate or treat one or more additional symptoms of a disease ordisorder discussed herein.

Accordingly, in one aspect, the compounds of the invention can be usedto treat pain, including acute, chronic, malignant and non-malignantsomatic pain (including cutaneous pain and deep somatic pain), visceralpain, and neuropathic pain. It is further understood that the compoundscan also alleviate or treat one or more additional signs or symptoms ofpain and sensory deficits (e.g., hyperalgesia, allodynia, dysesthesia,hyperesthesia, hyperpathia, paresthesia).

In some embodiments of this aspect of the invention, the compounds ofthe invention can be used to treat somatic or cutaneous pain associatedwith injuries, inflammation, diseases and disorders of the skin andrelated organs including, but not limited to, cuts, burns, lacerations,punctures, incisions, surgical pain, post-operative pain, orodentalsurgery, psoriasis, eczema, dermatitis, and allergies. The compounds ofthe invention can also be used to treat somatic pain associated withmalignant and non-malignant neoplasm of the skin and related organs(e.g., melanoma, basal cell carcinoma).

In other embodiments of this aspect of the invention, the compounds ofthe invention can be used to treat deep somatic pain associated withinjuries, inflammation, diseases and disorders of the musculoskeletaland connective tissues including, but not limited to, arthralgias,myalgias, fibromyalgias, myofascial pain syndrome, dental pain, lowerback pain, pain during labor and delivery, surgical pain, post-operativepain, headaches, migraines, idiopathic pain disorder, sprains, bonefractures, bone injury, osteoporosis, severe burns, gout, arthritis,osteoarthithis, myositis, and dorsopathies (e.g., spondylolysis,subluxation, sciatica, and torticollis). The compounds of the inventioncan also be used to treat deep somatic pain associated with malignantand non-malignant neoplasm of the musculoskeletal and connective tissues(e.g., sarcomas, rhabdomyosarcomas, and bone cancer).

In other embodiments of this aspect of the invention, compounds of theinvention can be used to treat visceral pain associated with injuries,inflammation, diseases or disorders of the circulatory system, therespiratory system, the genitourinary system, the gastrointestinalsystem and the eye, ear, nose and throat.

For example, the compounds of the invention can be used to treatvisceral pain associated with injuries, inflammation and disorders ofthe circulatory system associated including, but are not limited to,ischaemic diseases, ischaemic heart diseases (e.g., angina pectoris,acute myocardial infarction, coronary thrombosis, coronaryinsufficiency), diseases of the blood and lymphatic vessels (e.g.,peripheral vascular disease, intermittent claudication, varicose veins,haemorrhoids, embolism or thrombosis of the veins, phlebitis,thrombophlebitis lymphadenitis, lymphangitis), and visceral painassociated with malignant and non-malignant neoplasm of the circulatorysystem (e.g., lymphomas, myelomas, Hodgkin's disease).

In another example, the compounds of the invention can be used to treatvisceral pain associated with injuries, inflammation, diseases anddisorders of the respiratory system including, but are not limited to,upper respiratory infections (e.g., nasopharyngitis, sinusitis, andrhinitis), influenza, pneumoniae (e.g., bacterial, viral, parasitic andfungal), lower respiratory infections (e.g., bronchitis, bronchiolitis,tracheobronchitis), interstitial lung disease, emphysema,bronchiectasis, status asthmaticus, asthma, pulmonary fibrosis, chronicobstructive pulmonary diseases (COPD), diseases of the pleura, andvisceral pain associated with malignant and non-malignant neoplasm ofthe respiratory system (e.g., small cell carcinoma, lung cancer,neoplasm of the trachea, of the larynx).

In another example, the compounds of the invention can be used to treatvisceral pain associated with injuries, inflammation and disorders ofthe gastrointestinal system including, but are not limited to, injuries,inflammation and disorders of the tooth and oral mucosa (e.g., impactedteeth, dental caries, periodontal disease, oral aphthae, pulpitis,gingivitis, periodontitis, and stomatitis), of the oesophagus, stomachand duodenum (e.g., ulcers, dyspepsia, oesophagitis, gastritis,duodenitis, diverticulitis and appendicitis), of the intestines (e.g.,Crohn's disease, paralytic ileus, intestinal obstruction, irritablebowel syndrome, neurogenic bowel, megacolon, inflammatory bowel disease,ulcerative colitis, and gastroenteritis), of the peritoneum (e.g.peritonitis), of the liver (e.g., hepatitis, liver necrosis, infarctionof liver, hepatic veno-occlusive diseases), of the gallbladder, biliarytract and pancreas (e.g., cholelithiasis, cholecystolithiasis,choledocholithiasis, cholecystitis, and pancreatitis), functionalabdominal pain syndrome (FAPS), gastrointestinal motility disorders, aswell as visceral pain associated with malignant and non-malignantneoplasm of the gastrointestinal system (e.g., neoplasm of theoesophagus, stomach, small intestine, colon, liver and pancreas).

In another example, the compounds of the invention can be used to treatvisceral pain associated with injuries, inflammation, diseases, anddisorders of the genitourinary system including, but are not limited to,injuries, inflammation and disorders of the kidneys (e.g.,nephrolithiasis, glomerulonephritis, nephritis, interstitial nephritis,pyelitis, pyelonephritis), of the urinay tract (e.g. includeurolithiasis, urethritis, urinary tract infections), of the bladder(e.g. cystitis, neuropathic bladder, neurogenic bladder dysfunction,overactive bladder, bladder-neck obstruction), of the male genitalorgans (e.g., prostatitis, orchitis and epididymitis), of the femalegenital organs (e.g., inflammatory pelvic disease, endometriosis,dysmenorrhea, ovarian cysts), as well as pain associated with malignantand non-malignant neoplasm of the genitourinary system (e.g., neoplasmof the bladder, the prostate, the breast, the ovaries).

In further embodiments of this aspect of the invention, compounds of theinvention can be used to treat neuropathic pain associated withinjuries, inflammation, diseases and disorders of the nervous system,including the central nervous system and the peripheral nervous systems.Examples of such injuries, inflammation, diseases or disordersassociated with neuropathic pain include, but are not limited to,neuropathy (e.g., diabetic neuropathy, drug-induced neuropathy,radiotherapy-induced neuropathy), neuritis, radiculopathy, radiculitis,neurodegenerative diseases (e.g., muscular dystrophy), spinal cordinjury, peripheral nerve injury, nerve injury associated with cancer,Morton's neuroma, headache (e.g., nonorganic chronic headache,tension-type headache, cluster headache and migraine), migraine,multiple somatization syndrome, postherpetic neuralgia (shingles),trigeminal neuralgia complex regional pain syndrome (also known ascausalgia or Reflex Sympathetic Dystrophy), radiculalgia, phantom limbpain, chronic cephalic pain, nerve trunk pain, somatoform pain disorder,central pain, non-cardiac chest pain, central post-stroke pain.

In another aspect, the compounds of the invention can be used to treatinflammation associated with injuries, diseases or disorders of the skinand related organs, the musculoskeletal and connective tissue system,the respiratory system, the circulatory system, the genitourinary systemand the gastrointestinal system.

In some embodiments of this aspect of the invention, examples ofinflammatory conditions, diseases or disorders of the skin and relatedorgans that can be treated with the compounds of the invention include,but are not limited to allergies, atopic dermatitis, psoriasis anddermatitis.

In other embodiments of this aspect of the invention, inflammatoryconditions, diseases or disorders of the musculoskeletal and connectivetissue system that can be treated with the compounds of the inventioninclude, but are not limited to arthritis, osteoarthritis, and myositis.

In other embodiments of this aspect of the invention, inflammatoryconditions, diseases or disorders of the respiratory system that can betreated with the compounds of the invention include, but are not limitedto allergies, asthma, rhinitis, neurogenic inflammation, pulmonaryfibrosis, chronic obstructive pulmonary disease (COPD), adultrespiratory distress syndrome, nasopharyngitis, sinusitis, andbronchitis.

In still other embodiments of this aspect of the invention, inflammatoryconditions, disease or disorders of the circulatory system that can betreated with the compounds of the invention include, but are not limitedto, endocarditis, pericarditis, myocarditis, phlebitis, lymphadenitisand artherosclerosis.

In further embodiments of this aspect of the invention, inflammatoryconditions, diseases or disorders of the genitourinary system that canbe treated with the compounds of the invention include, but are notlimited to, inflammation of the kidney (e.g., nephritis, interstitialnephritis), of the bladder (e.g., cystitis), of the urethra (e.g.,urethritis), of the male genital organs (e.g., prostatitis), and of thefemale genital organs (e.g., inflammatory pelvic disease).

In further embodiments of this aspect of the invention, inflammatoryconditions, diseases or disorders of the gastrointestinal system thatcan be treated with the compounds of the invention include, but are notlimited to, gastritis, gastroenteritis, colitis (e.g., ulcerativecolitis), inflammatory bowel syndrome, Crohn's disease, cholecystitis,pancreatitis and appendicitis.

In still further embodiments of this aspect of the invention,inflammatory conditions, diseases or disorders that can be treated withthe compounds of the invention, but are not limited to inflammationassociated with microbial infections (e.g., bacterial, viral and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions and autoimmune diseases(e.g., lupus erythematosus).

In another aspect, the compounds of the invention can be used to treatinjuries, diseases or disorders of the nervous system including, but notlimited to neurodegenerative diseases (e.g., Alzheimer's disease,Duchenne's disease), epilepsy, multiple sclerosis, amyotrophic lateralsclerosis, stroke, cerebral ischemia, neuropathies (e.g.,chemotherapy-induced neuropathy, diabetic neuropathy), retinal pigmentdegeneration, trauma of the central nervous system (e.g., spinal cordinjury), and cancer of the nervous system (e.g., neuroblastoma,retinoblastoma, brain cancer, and glioma), and other certain cancers(e.g., melanoma, pancreatic cancer).

In further aspects of the invention, the compounds of the invention canalso be used to treat other disorders of the skin and related organs(e.g., hair loss), of the circulatory system, (e.g., cardiac arrhythmiasand fibrillation and sympathetic hyper-innervation), and of thegenitourinary system (e.g., neurogenic bladder dysfunction andoveractive bladder).

The present invention provides a method for treating a subject thatwould benefit from administration of a composition of the presentinvention. Any therapeutic indication that would benefit from a gatedion channel modulator can be treated by the methods of the invention.The method includes the step of administering to the subject acomposition of the invention, such that the disease or disorder istreated.

The invention further provides a method for preventing in a subject, adisease or disorder which can be treated with administration of thecompositions of the invention. Subjects “at risk” may or may not havedetectable disease, and may or may not have displayed detectable diseaseprior to the treatment methods described herein. “At risk” denotes thatan individual who is determined to be more likely to develop a symptombased on conventional risk assessment methods or has one or more riskfactors that correlate with development of a disease or disorder thatcan be treated to according the methods of the invention. For example,risk factors include family history, medication history, and history ofexposure to an environmental substance which is known or suspected toincrease the risk of disease. Subjects at risk for a disease orcondition which can be treated with the agents mentioned herein can alsobe identified by, for example, any or a combination of diagnostic orprognostic assays known to those skilled in the art. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the disease or disorder, such that the disease ordisorder is prevented or, alternatively, delayed in its progression.

EXEMPLIFICATION OF THE INVENTION

The invention is further illustrated by the following examples, whichcould be used to examine the gated ion channel modulating activity ofthe compounds of the invention. The example should not be construed asfurther limiting. The animal models used throughout the Examples areaccepted animal models and the demonstration of efficacy in these animalmodels is predictive of efficacy in humans.

Example 1 Identification of ASIC Antagonists Using Calcium-Imaging CellCulture

ASIC1a expressing HEK293 cells are grown in culture medium (DMEM with10% FBS), in polystyrene culture flasks (175 mm²) at 37° C. in ahumidified atmosphere of 5% CO₂. Confluency of cells should be 80-90% onday of plating. Cells are rinsed with 10 ml of PBS and re-suspended byaddition of culture medium and trituration with a 25 ml pipette.

The cells are seeded at a density of approximately 1×10⁶ cells/ml (100μl/well) in black-walled, clear bottom, 96-well plates pre-treated with10 mg/l poly-D-lysin (75 μl/well for ≧30 min). Plated cells were allowedto proliferate for 24 h before loading with dye.

Loading with Fluorescent Calcium Dye Fluo-4/AM

Fluo-4/AM (1 mg, Molecular Probes) is dissolved in 912 μl DMSO. TheFluo-4/AM stock solution (1 mM) is diluted with culture medium to afinal concentration of 2 μM (loading solution).

The culture medium is aspirated from the wells, and 50 μl of theFluo-4/AM loading solution is added to each well. The cells areincubated at 37° C. for 30 min.

Calcium Measurements

After the loading period, the loading solution is aspirated and thecells are washed twice with 100 μl modified Assay Buffer (145 mM NaCl, 5mM KCl, 5 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4) to removeextracellular dye. Following the second wash, 100 μl modified AssayBuffer is added to each well and the fluorescence is measured in FLIPR™or FlexStatiomm (Molecular Devices, USA), or any other suitableequipment known to the skilled in the art.

FLIPR Settings (ASIC1a)

Temperature: Room temperature (20-22° C.)

First addition: 50 μl test solution at a rate of 30 pd/sec and astarting height of 100 μl

Second addition: 50 μl MES solution (20 mM, 5 mM final concentration) ata rate of 35 μl/sec and a starting height of 150

Reading intervals: pre-incubation −10 sec×7 and 3 sec×3 antagonist phase3 sec×17 and 10 sec×12

Addition plates (compound test plate and MES plate) are placed on theright and left positions in the FLIPR tray, respectively. Cell platesare placed in the middle position and the ASIC1a program is effectuated.FLIPR will then take the appropriate measurements in accordance with theinterval settings above. Fluorescence obtained after stimulation iscorrected for the mean basal fluorescence (in modified Assay Buffer).

Hit Confirmation and Characterization of Active Substances

The MES-induced peak calcium response, in the presence of testsubstance, is expressed relatively to the MES response alone. Testsubstances that block the MES-induced calcium response are re-tested intriplicates. Confirmed hits are picked for further characterization byperforming full dose-response curves to determine potency of each hitcompound as represented by the IC₅₀ values (i.e., the concentration ofthe test substance which inhibits 50% of the MES-induced calciumresponse; see, for example, FIGS. 1A, 1B and 1C, and FIGS. 13A and 13B).

A summary of IC₅₀ values of compounds of the invention as acquired inthe calcium mobilization assay are shown below. n=3−7

Compound IC₅₀ (μM) Compound A 0.10-0.20 Compound B 0.020-0.030 CompoundC 0.20-0.30 Compound D 0.250-0.350 Compound H 0.015-0.25  Compound J0.1-0.7 Compound K 0.3-1.7

Example 2 Screening and Bioanalysis of ASIC Antagonists in HeterologousExpression Systems

This example describes another in vitro assessment of the activity ofthe compounds of the present invention.

Another example of an in vitro assessment method consists of usingmammalian heterologous expression systems, which are known to theskilled in the art, and include a variety of mammalian cell lines suchas COS, HEK, e.g., HEK293 and/or CHO, cells. Cell lines are transfectedwith gated ion channel(s) and used to perform electrophysiology asfollows:

All experiments are performed at room temperature (20-25° C.) in voltageclamp using conventional whole cell patch clamp methods (Neher, E., etal. (1978) Pfluegers Arch 375:219-228).

The amplifier used is the EPC-9 (HEKA-electronics, Lambrect, Germany)run by a Macintosh G3 computer via an ITC-16 interface. Experimentalconditions are set with the Pulse-software accompanying the amplifier.Data is low pass filtered and sampled directly to hard-disk at a rate of3 times the cut-off frequency.

Pipettes are pulled from borosilicate glass using a horizontal electrodepuller (Zeitz-Instrumente, Augsburg, Germany). The pipette resistancesare 2-3 MOhms in the salt solutions used in these experiments. Thepipette electrode is a chloridized silver wire, and the reference is asilver chloride pellet electrode (In Vivo Metric, Healdsburg, USA) fixedto the experimental chamber. The electrodes are zeroed with the openpipette in the bath just prior to sealing.

Coverslips with the cells are transferred to a 15 μl experimentalchamber mounted on the stage of an inverted microscope (IMT-2, Olympus)supplied with Nomarski optics. Cells are continuously superfused withextracellular saline at a rate of 2.5 ml/min. After giga-seal formation,the whole cell configuration is attained by suction. The cells are heldat a holding voltage of −60 mV and at the start of each experiment thecurrent is continuously measured for 45 s to ensure a stable baseline.Solutions of low pH (<7) are delivered to the chamber through acustom-made gravity-driven flowpipe, the tip of which is placedapproximately 50 μm from the cell. Application is triggered when thetubing connected to the flowpipe is compressed by a valve controlled bythe Pulse-software. Initially, low pH (in general, pH 6.5) is appliedfor 5 s every 60 s. The sample interval during application is 550 μs.After stable responses are obtained, the extracellular saline as well asthe low-pH solution are switched to solutions containing the compound tobe tested. The compound is present until responses of a repeatableamplitude are achieved. Current amplitudes are measured at the peak ofthe responses, and effect of the compounds is calculated as theamplitude at compound equilibrium divided by the amplitude of thecurrent evoked by the pulse just before the compound was included.

The following salt solutions are used: extracellular solution (mM): NaCl(140), KCl (4), CaCl₂ (2), MgCl₂ (4), HEPES (10, pH 7.4); intracellularsolution (mM): KCl (120), KOH (31), MgCl₂ (1.785), EGTA (10), HEPES (10,pH 7.2). In general, compounds for testing are dissolved in 50% DMSO at500 fold the highest concentration used.

Patch Clamp experiments with Compound A and Compound H demonstrated theefficacy to inhibit recombinant rat ASIC gated channels as illustratedin FIGS. 2A and 2B. HEK293 cells were transfected with rASIC1a or rASIC3and were used to perform full dose-inhibition curves with Compound A,Compound H and amiloride. Results are expressed as a fraction of thecontrol peak current obtained in the absence of the test substance.These data indicate that both Compounds A and H are more potentantagonists as compared to amiloride.

Similar findings are shown in FIG. 3 with the human ASIC1a stablytransfected in CHO cells. FIG. 3A compares the dose-responserelationship between Compound A and amiloride [determined by measuringthe area under the curve of the response (total charge transfer) andnormalized to the control response]. Both Compound A (FIG. 3B) andamiloride were able to reduce the human ASIC1a pH-evoked response in adose-dependent manner. However, Compound A was about 100 fold morepotent. FIGS. 14 A and 14B show similar results for Compounds J and K.

FIGS. 4A and 4B show response to acidic saline in the absence orpresence of 10 μM of Compound A recorded from HEK293 cells expressingeither hASIC1a alone or HEK293 cells co-expressing hASIC1a and hASIC3[voltage clamped at −60 mV, extracellular solution surrounding cells waschanged rapidly from pH 7.4 to pH 6.5 for 5 sec] (n=3). The data shownin these figures demonstrate that Compound A effectively modulates theactivity of these gated ion channels.

Example 3 Screening and Bioanalysis of ASIC Antagonists in Xenopuslaevis Oocytes

This example describes the in vitro assessment of the activity of thecompounds of the present invention.

Two-electrode voltage clamp electrophysiological assays in Xenopuslaevis oocytes expressing gated ion channels are performed as follows:

Oocytes are surgically removed from adult Xenopus laevis and treated for2 h at room temperature with 1 mg/ml type I collagenase (Sigma) inBarth's solution under mild agitation. Selected oocytes at stage IV-Vare defolliculated manually before nuclear microinjection of 2.5-5 ng ofa suitable expression vector, such as pcDNA3, comprising the nucleotidesequence encoding a gated ion channel subunit protein. In such anexperiment, the oocytes express homomultimeric proton-gated ion channelson their surface. In an alternate experiment, one, two, three or morevectors comprising the coding sequences for distinct gated ion channelsubunits are co-injected in the oocyte nuclei. In the latter case,oocytes express heteromultimeric proton-gated ion channels. For example,ASIC2a and/or ASIC3 subunits in pcDNA3 vector are co-injected at a 1:1cDNA ratio. After 2-4 days of expression at 19° C. in Barth's solutioncontaining 50 mg/ml gentamicin and 1.8 mM CaCl₂, gated ion channels areactivated by applying an acidic solution (pH<7) and currents arerecorded in a two electrode voltage-clamp configuration, using anOC-725B amplifier (Warner Instruments). Currents are acquired anddigitized at 500 Hz on an Apple Imac G3 computer with an A/D NB-MIO-16XLinterface (National Instruments) and recorded traces are post-filteredat 100 Hz in Axograph (Axon Instruments) (Neher, E. and Sakmann, B.(1976) Nature 260:799-802). Once impaled with the microelectrodes,oocytes are continuously superfused at 10-12 ml/min with a modifiedRinger's solution containing 97 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, and 10mM HEPES brought to pH 7.4 with NaOH (Control Ringer). Test Ringersolution is prepared by replacing HEPES with MES and adjusting the pH tothe desired acidic value. Compounds of the present invention areprepared in both the Control and Test Ringer solutions and applied tooocytes at room temperature through a computer-controlled switchingvalve system. Osmolarity of all solutions is adjusted to 235 mOsm withcholine chloride. Similarly, recordings can also be acquired in anautomated multichannel oocytes system as the OpusExpress™ (MolecularDevices, Sunnyvale, USA).

FIGS. 5A and 5B show the inward currents elicited by the application ofa pH 6.5 test ringer solution in the presence and absence of Compound Aat 30 μM in an OpusExpress™ system. Recordings were acquired fromoocytes expressing homomeric hASIC1a (FIG. 5A) or heteromeric hASIC1a+3(FIG. 5B) using a two electrode voltage-clamp configuration procedure asdescribed herein. Data shown in these figures demonstrate that CompoundA effectively modulates the activity of these gated ion channels.

Example 4 Screening and Bioanalysis of ASIC Antagonists in Primary CellSystems

This example describes another in vitro assessment of the inhibitoryactivity of the compounds of the present invention utilizing patch-clampelectrophysiology of sensory neurons in primary culture.

Sensory neurons can be isolated and cultured in vitro from differentanimal species. The most widely used protocols use sensory neuronsisolated from neonatal (Eckert, et al. (1997) J Neurosci Methods77:183-190) and embryonic (Vasko, et al. (1994) J Neurosci 14:4987-4997)rat. Trigeminal and dorsal root ganglion sensory neurons in cultureexhibit certain characteristics of sensory neurons in vivo.Electrophysiology is performed similarly as described above in Example2. In the voltage-clamp mode, trans-membrane currents are recorded, asshown in FIGS. 6A and 6B where Compounds A and H at 1 μM inhibit the pH6.5-induced inward current. In the current-clamp mode, change in thetrans-membrane potential are recorded. Under acidic conditions (e.g., pH6.5) the membrane depolarizes, leadin to the firing of actionpotentials, as shown in FIGS. 7A and 7B. Compounds A and H at 1 μMinhibit the acid-induced membrane depolarization and reduces the ensuingrate of action potential firing. Data shown in these figures demonstratethat Compounds A and H effectively modulate the activity of these nativesensory-neuron gated ion channels (n=3).

Example 5 Formalin Model—Model Of Acute Tonic Pain

This example describes the in vivo assessment of the inhibitory activityof the compounds of the present invention.

A number of well-established models of pain are described in theliterature and are known to the skilled in the art (see, for example,Table 1). This example describes the use of the Formalin test.

Male Sprague-Dawley rats are housed together in groups of three animalsunder standard conditions with unrestricted access to food and water.All experiments are conducted according to the ethical guidelines forinvestigations of experimental pain in conscious animals (Zimmerman,1983)

Assessment of formalin-induced flinching behavior in normal, uninjuredrats (body weight 150-180 g) was made with the use of an AutomatedNociception Analyser (University of California, San Diego, USA).Briefly, this involved placing a small C-shaped metal band (10 mmwide×27 mm long) on the hindpaw of the rat to be tested. The rats (fourrats were included in each testing session) were then placed in acylindrical plexiglass observation chamber (diameter 30.5 cm and height15 cm) for 20 min for adaptation purposes prior to being administereddrug or vehicle according to the experimental paradigm being followed.After adaptation, individual rats were then gently restrained andformalin (5% in saline, 50 s.c.) was injected into the plantar surfaceof the hindpaw using a 27G needle. Rats were then returned to theirseparate observation chambers, each of which were in turn situated uponan enclosed detection device consisting of two electromagnetic coilsdesigned to produce an electromagnetic field in which movement of themetal band could be detected. The analogue signal was then digitised anda software algorithm (LabView) applied to enable discrimination offlinching behaviour from other paw movements. A sampling interval of 1min was used and on the basis of the resulting response patterns 5phases of nociceptive behaviour were identified and scored: first phase(P1; 0-5 min), interphase (Int; 6-15 min), second phase (P2; 60 min),phase 2A (P2A; 16-40 min) and phase 2B (P2B; 41-60 min).

Nociceptive behavior was also determined manually every 5 min bymeasuring the amount of time spent in each of four behavioralcategories: 0, treatment of the injected hindpaw is indistinguishablefrom that of the contralateral paw; 1, the injected paw has little or noweight placed on it; 2, the injected paw is elevated and is not incontact with any surface; 3, the injected paw is licked, bitten, orshaken. A weighted nociceptive score, ranging from 0 to 3 was calculatedby multiplying the time spent in each category by the category weight,summing these products, and dividing by the total time for each 5 minblock of time. (Coderre et al., Pain 1993; 54: 43). On the basis of theresulting response patterns, 2 phases of nociceptive behavior wereidentified and scored: first phase (P1; 0-5 min), interphase (Int; 6-15min), second phase (P2; 60 min), phase 2A (P2A; 16-40 min) and phase 2B(P2B; 41-60 min).

Statistical analysis was performed using Prism™ 4.01 software package(GraphPad, San Diego, Calif., USA). The difference in response levelsbetween treatment groups and control vehicle group was analyzed using anANOVA followed by Bonferroni's method for post-hoc pair-wisecomparisons. A p value <0.05 was considered to be significant

FIGS. 8 and 9 are representative examples of the effect of Compound A onpain induced by intraplantar formalin injection. Compound A wasadministered i.p. 30 min. before the formalin. Compound A was able toreduce the total pain score behavior (flinching, licking, biting) (FIG.8A) in phase 1 and 2 of the formalin test. These effects on both phases1 and 2 were quite pronounced when only specific pain behaviors such asliking and biting were observed (FIG. 8B) (n=6-8). The results formthese experiments are summarized in FIG. 9, where a clear dose-responserelationship for the Phase 2 of the total pain score can be seen (FIG.9A) with an ED₅₀ of about 12 mg/kg. In these experiments, a linearrelationship between dose and plasma exposure was observed (FIG. 9B).Similar results are shown for compound B and H (FIGS. 10A, and B) usingthe Automate Nociceptive Analyzer described above (n=6-8). These resultsindicate that Compounds A, B and H can block acute tonic pain induced byformalin injection in the paw.

Example 6 CFA Model—Model of Chronic Inflammatory Pain

Injection of complete Freunds adjuvant (CFA) in the hindpaw of the rathas been shown to produce a long-lasting inflammatory condition, whichis associated with behavioural hyperalgesia and allodynia at theinjection site (Hylden et al., Pain 37: 229-243, 1989) (Blackburn-Munroet al., 2002). Rats (body weight 260-300 g) were given a s.c. injectionof CFA (50% in saline, 100 Sigma) into the plantar surface of thehindpaw under brief halothane anaesthesia. After 24 h, they were thentested for hindpaw weight bearing responses, as assessed using anIncapacitance Tester (Linton Instrumentation, UK), (Zhu et al., 2005).The instrument incorporates a dual channel scale that separatelymeasures the weight of the animal distributed to each hindpaw. Whilenormal rats distribute their body weight equally between the twohindpaws (50-50), the discrepancy of weight distribution between aninjured and non-injured paw is a natural reflection of the discomfortlevel in the injured paw (nocifensive behavior). The rats were placed inthe plastic chamber designed so that each hindpaw rested on a separatetransducer pad. The averager was set to record the load on thetransducer over 5 s time period and two numbers displayed representedthe distribution of the rat's body weight on each paw in grams (g). Foreach rat, three readings from each paw were taken and then averaged.Side-to-side weight bearing difference was calculated as the average ofthe absolute value of the difference between two hindpaws from threetrials (right paw reading-left paw reading).

Assessment of thermal hyperalgesia: Baseline and post-treatmentwithdrawal latencies to a noxious thermal stimulus were measuredaccording to Hargreaves (Hargreaves et al., 1988) using a plantar testanalgesia meter (IITC, Woodland Hills, Calif., model #336). The stimulusintensity was set at 30% of maximum output and the cut-off time was setat 30 seconds. Rats were placed on a glass plate warmed to 28° C. andallowed to habituate to the testing chambers for a minimum of 15 minutesprior to each testing session. The thermal stimulus was applied to theplantar surface of the paw, and the mean latency of three readings oneach paw was used as the latency value for each time point. Thermalthresholds were defined as the latency in seconds to the first painbehavior, which includes nocifensive paw withdrawal, flinching, bitingand/or licking of the stimulated paw. The mean and standard error of themean (SEM) were determined for the injured and normal paws for eachtreatment group.

FIGS. 11A, 11B and 11C demonstrate the effect of Compound A, Compound Hand morphine on spontaneous pain behaviours in CFA-treated rats. Hindpawweight bearing responses were measured in male Sprague-Dawley rats for2-3 days prior to being given a hindpaw injection of CFA. Twenty-fourhours later baseline responses were measured and rats were thenadministered Compound A (5, 10 and 20 mg/kg, i.p.), Compound H (10, 30and 60 mg/kg) and morphine (3, 6 and 10 mg/kg). Weight bearing responseswere then measured at 30, 60 and 120 min after drug or vehicle injection(data shown at 60 min.). Compounds A and H as well as morphine produceda marked dose-dependent attenuation in the CFA-induced change in weightbearing compared with vehicle. Data are expressed as mean+/−SEM.*P<0.05, **P<0.01, ***P<0.001 vs baseline; +++: P<0.001 vs vehicle. Allgroups n=7-8.

FIG. 12 depicts the dose-dependent reversal of the CFA-induced thermalhyperalgesia by Compound A. CFA was injected 48 h prior to testing ofCompound A. Thermal hyperalgesia was measured 3 h after i.p.administration of Compound A. Compound A was capable of fully reversingthe thermal hyperalgesia with an ED₅₀ of 6.5 mg/kg. For comparison,results with morphine (6 mg/kg sc) and indomethacin (30 mg/kg po) areshown. These results demonstrate that Compounds A is efficacious in bothmechanical and thermal modalities. Data are expressed as mean±SEM,(n=10). **P<0.01, vs. baseline.

Example 7 Cloning and Expression of ASICs

The cDNA for ASIC1a and ASIC3 can be cloned from rat poly(A)⁺mRNA andput into expression vectors according to Hesselager et al. (J Biol.Chem. 279(12):11006-15 2004). All constructs are expressed in CHO-K¹cells (ATCC no. CCL61) or HEK293 cells. CHO-K¹ cells are cultured at 37°C. in a humidified atmosphere of 5% CO₂ and 95% air and passaged twiceevery week. The cells are maintained in DMEM (10 mM HEPES, 2 mMglutamax) supplemented with 10% fetal bovine serum and 2 mM L-proline(Life Technologies). CHO-K1 cells are co-transfected with plasmidscontaining ASICs and a plasmid encoding enhanced green fluorescentprotein (EGFP) using the lipofectamine PLUS transfection kit (LifeTechnologies) or Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol. For each transfection it is attempted to use anamount of DNA that yield whole-cell currents within a reasonable range(0.5 nA-10 nA), in order to avoid saturation of the patch-clampamplifier (approximately 50 ng for ASIC 1a and ASIC3).Electrophysiological measurements are performed 16-48 hours aftertransfection. The cells are trypsinized and seeded on glass coverslipsprecoated with poly-D-lysine, on the day the electrophysiologicalrecordings were performed.

Example 8 Synthetic Procedure for Compound A 5-Bromo-8-nitroisoquinoline(II)

5-Bromo-8-nitroisoquinoline was prepared from the correspondingisoquinoline (I) according to the procedure found in William Dalby Brownand Alex Haahr Gouliaev, Organic Syntheses Vol. 81, p 98.

5-Bromo-1,2,3,4-tetrahydro-2-methyl-8-nitroisoquinoline (III)

5-Bromo-8-nitroisoquinoline (II, 5 g, 19.7 mmol) was suspended inanhydrous DMF (20 mL) under nitrogen atmosphere and the mixture washeated until the isoquinoline was dissolved completely.Methyl-p-toluenesulphonate (4 g, 21.5 mmol) was added dropwise,whereafter heated at 85° C. for 24 hours. After cooling in an ice bath,the solid was collected by filtration and washed with ether and acetoneto give the isoquinolinium salt (used without further purification).

The isoquinolinium salt was dissolved in acetic acid (30 ml) and sodiumborohydride (0.87 g) was added. The reaction mixture was stirred at roomtemperature overnight. The acetic acid was removed under vacuum and thendiluted with water. The solution was basified with 10N NaOH (pH=8) andthe precipitated product was collected by filtration, washed with waterand dried under vacuum to give light sensitive5-bromo-1,2,3,4-tetrahydro-2-methyl-8-nitroisoquinoline (4.7 g).

5-Bromo-1,2,3,4-tetrahydro-2-methylisoquinolin-8-amine (IV)

To a solution of N-methyl-5-bromo-8-nitro-1,2,3,4-tetrahydroisoquinoline(III, 4.7 gm, 17.3 mmol) in ethanol (50 ml), Raney Nickel (solution inwater, 1.5 g) was added. The reaction mixture was stirred at roomtemperature overnight under H₂. The mixture was filtered through celiteand solvent was removed under vacuum to give IV.

N-(5-Bromo-1,2,3,4-tetrahydro-2-methylisoquinolin-8-yl)-2-(hydroxyimino)acetamide(V)

A mixture of 5-bromo-1,2,3,4-tetrahydro-2-methylisoquinolin-8-amine (IV,3.25 g, 13.5 mmol), chloral hydrate (2.3 g), hydroxylamine hydrochloride(2.9 g), 12 g Na₂SO₄ (12 g) in H₂O:EtOH (3:1, 50 mL) was refluxed for 1hr whereafter it was cooled to 60° C. and carefully basified with 4NNaOH to pH=7 and allowed to cool. The solid was collected by filtration,washed with water and dried under vacuum to give V.

5-Bromo-6,7,8,9-tetrahydro-8-methyl-1H-pyrrolo[3,2,-h]isoquinoline-2,3-dione(VI)

To preheated sulphuric acid (20 mL, 70° C.),N-(5-bromo-1,2,3,4-tetrahydro-2-methylisoquinolin-8-yl-2-(hydroxyimino)acetamide(V, 3.5 g) was added portion-wise over a period of 30 min. The heatingwas continued further for 1 hr. The reaction mixture was cooled to roomtemperature and quenched by pouring over ice cold water (100 mL) andthen neutralized with aqueous 10N NaOH. The precipitated product wasfiltered, washed with water to give isatin VI.

5-Bromo-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2,-h]isoquinoline-2(3H)-one(VII)

To the solution of isatin VI (3.5 g) in methanol (50 ml), hydroxylaminehydrochloride (2.0 g) was added and mixture was refluxed 1 hr. Thereaction mixture was cooled to room temperature and solid was collectedby filtration, washed with ethanol and ether and dried under vacuum.

Compound A

A mixture of5-bromo-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinolin-2(3H)-one(VII, 100 mg), 5-fluoro-2-methoxyphenylboronic acid (60 mg), potassiumphosphate (72 mg), dichlorobis(triphenylphosphine)palladium(II) (11 mg),water (1.5 mL) and DMF (3 mL) was irradiated under Microwave (120° C.,10 min). The solvent was evaporated under vacuum and residue waschromatographed on silica gel to give5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2,-h]isoquinolin-2(3H)-one.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

1. A method of treating a clinical condition selected from the groupconsisting of inflammatory disorders, a disease or disorder associatedwith the genitourinary system, a disease or disorder associated with thegastrointestinal system, a disease or disorder of the musculoskeletaltissue and a disease or disorder of the connective tissue in a subjectin need thereof comprising administering to the subject a compoundrepresented by formula 1,

or pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof; wherein thedashed lines indicate a single or double bond; and R¹ is selected fromthe group consisting of hydrogen, alkyl, alkoxy-alkyl, hydroxy-alkyl,alkoxy-carbonyl-alkyl, alkyl-carbonyl-oxy-alkyl, cycloalkyl,cycloalkyl-alkyl, alkenyl, alkynyl, alkoxy, sulfonamide, amino,sulfonyl, sulfonic acid, urea, phenyl and benzyl, in which the phenyl orbenzyl group is optionally substituted with one or more selected fromthe group consisting of halogen, CF₃, nitro, amino, cyano,hydroxy-alkyl, alkoxy, sulfonamide, alkenyl, alkynyl, amino, sulfonyl,sulfonic acid and urea; R² is selected from the group consisting ofhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, —(CH₂)₁₋₄S(O)₃H,—C(O)C₁₋₄alkyl and —S(O)₂C₁₋₄alkyl; R³ is selected from the groupconsisting of hydrogen, hydroxyl, alkyl, acyl, phenyl, benzyl, —COON,—C(O)N(CH₃)₂, —O-phenyl, —OCF₃, alkoxy, —O(CH₂)₀₋₄OCH₃, —C(O)H,—C(O)CH₃,

and R⁴ and R⁵ are each, independently, selected from the groupconsisting of halogen, CF₃, nitro, amino, cyano, hydroxyl, alkyl,alkoxy, phenoxy, phenyl and —SO₂NR′R″, wherein R′ and R″ independentlyof each another represents hydrogen or alkyl.
 2. The method of claim 1,wherein the compound is represented by the Formula 2,

or pharmaceutically acceptable salts, enantiomers, stereoisomers,rotamers, tautomers, diastereomers, or racemates thereof; wherein R¹ isselected from the group consisting of hydrogen, C₁₋₄-alkyl,C₁₋₄-alkenyl, and C₁₋₄-alkynyl; R² is selected from the group consistingof hydrogen, hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkenyl and C₁₋₄-alkynyl; and R⁴and R⁵ are each, independently, selected from the group consisting ofhalogen, CF₃, nitro, amino, cyano, hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkoxy,phenoxy and phenyl.
 3. The method of claim 1, wherein the compound isrepresented by the Formula 3,

or a pharmaceutically acceptable salt thereof, wherein R¹ is selectedfrom the group consisting of hydrogen, alkyl, alkoxy-alkyl,alkoxy-carbonyl-alkyl, alkyl-carbonyl-oxy-alkyl, cycloalkyl,cycloalkyl-alkyl, alkenyl, alkynyl, alkoxy, sulfonamide, amino,sulfonyl, sulfonic acid, urea phenyl and benzyl, in which the phenyl orbenzyl group is optionally substituted with one or more selected fromthe group consisting of halogen, CF₃, nitro, amino, cyano,hydroxy-alkyl, alkoxy, sulfonamide, alkenyl, alkynyl, amino, sulfonyl,sulfonic acid and urea; and R⁴ and R⁵ are each, independently, selectedfrom the group consisting of halogen, phenoxy, CF₃, nitro, amino, cyano,hydroxyl, alkyl, alkoxy, phenyl and SO₂NR′R″, wherein R′ and R″independently of each another represents hydrogen or alkyl.
 4. Themethod of claim 3, wherein R⁵ is in the 2 position of the aryl ring andR⁴ is in the 5 position of the aryl ring, or R⁴ is in the 3 position ofthe aryl ring and R⁵ is in the 5 position of the aryl ring.
 5. Themethod according to claim 4, wherein R⁴ and R⁵ each independently areselected from the group consisting of halogen, CF₃, nitro, amino, cyano,hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkoxy, phenoxy and phenyl.
 6. The method ofclaim 3, wherein R¹ is selected from the group consisting of hydrogen,C₁₋₄-alkyl, C₁₋₄-alkenyl, and C₁₋₄ alkynyl.
 7. The method of claim 3,wherein R¹ is selected from the group consisting of hydrogen,C₁₋₄-alkyl, C₁₋₄-alkenyl, and C₁₋₄-alkynyl and R⁴ and R⁵ are each,independently, selected from the group consisting of halogen, CF₃,nitro, amino, cyano, hydroxyl, C₁₋₄-alkyl, C₁₋₄-alkoxy, phenoxy andphenyl.
 8. The method of claim 3, wherein R¹ is selected from the groupconsisting of hydrogen and C₁₋₄-alkyl; and R⁴ and R⁵ are each,independently, selected from the group consisting of halogen, CF₃,C₁₋₄-alkyl, phenoxy and C₁₋₄-alkoxy.
 9. The method of claim 3, whereinR¹ is selected from the group consisting of C₁₋₄-alkyl; and R⁴ and R⁵are each, independently, selected from the group consisting of halogen,CF₃, C₁₋₄-alkyl, phenoxy and C₁₋₄-alkoxy.
 10. The method of claim 3,wherein R¹ is selected from the group consisting of —CH₃ and —CH₂CH₃;and R⁴ and R⁵ are each, independently, selected from the groupconsisting of halogen, phenoxy and C₁₋₄-alkoxy.
 11. The method of claim1, wherein the compound is selected from the group consisting of5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(3,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(3,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(2,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one;5-(2,3-dimethyl-phenyl)-8-ethyl-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]isoquinoline-2,3-dione3-oxime; and5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1H-pyrrolo[3,2-h]isoquinoline-2(3H)-one.12. The method of claim 1, wherein the disease or disorder is aninflammatory disorder of the genitourinary system and/or thegastrointestinal system.
 13. The method of claim 1, wherein the methodis a method for treating pain associated with a disease or disorder ofthe genitourinary system and/or the gastrointestinal system.
 14. Themethod of claim 12, wherein the inflammatory disorder of thegastrointestinal system is selected from the group consisting ofinflammatory bowel disorder, ulcerative colitis, Crohn's disease,diverticulitis, viral infection, bacterial infection, peptic ulcer,chronic hepatitis, gingivitis, periodentitis, stomatitis, gastritis andgastrointestinal reflux disease.
 15. The method of claim 1, wherein themethod is a method for treating visceral pain associated with disordersof the oesophagus, stomach, duodenum, colon and/or intestines.
 16. Themethod of claim 1, wherein the method is a method of treating visceralpain associated with a disorder selected from the group consisting ofulcers, dyspepsia, oesophagitis, gastritis, duodenitis, diverticulitis,appendicitis, Crohn's disease paralytic ileus, intestinal obstruction,irritable bowel syndrome, neurogene bowel, megacolon, inflammatory boweldisease, ulcerative colitis, gastroenteritis, functional abdominal painsyndrome and gastrointestinal motility disorders.
 17. The method ofclaim 1, wherein the disease or disorder is selected from the groupconsisting of gastritis, duodenitis, irritable bowel syndrome, colitis,Crohn's disease, gastrointestinal reflux disease, ulcers anddiverticulitis.
 18. The method of claim 1, wherein the clinicalcondition is a disease or disorder associated with the genitourinarysystem selected from the group consisting of inflammation of the kidney,inflammation of the bladder, inflammation of the urethra, inflammationof the male genital organs and inflammation of the female genitalorgans.
 19. The method of claim 1, wherein the method is a method oftreating deep somatic pain associated with a disease or disorder of themusculoskeletal tissue and a disease or disorder of the connectivetissue.
 20. The method according to claim 15, wherein the disease ordisorder is selected from the group consisting of arthralgias, myalgias,fibromyalgias, myofascial pain syndrome, dental pain, lower back pain,pain during labor and delivery, surgical pain, post-operative pain,headaches, migraines, idiopathic pain disorder, sprains, bone fractures,bone injury, osteoporosis, severe burns, gout, arthritis,osteoarthithis, myositis, and dorsopathies.
 21. The method according toclaim 1, wherein the clinical condition is an inflammatory conditionselected from the group consisting of arthritis, osteoarthritis andmyositis.